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444 preferred methods. These findings indicate that the adapted curriculum may improve dementia knowledge among RDs. FOSTERING AN UNDERSTANDING OF INTERPROFESSIONAL APPROACHES TO GERIATRICS Anna Faul, Pamela Yankeelov, and Sam Cotton, University of Louisville, Louisville, Kentucky, United States Serving older adults with multiple chronic conditions and variable social, emotional, or physical support effectively within the primary care setting requires an interdisciplinary approach to care, together with the integration of novel approaches to care coordination (Dorr et al, 2006). The purpose of this study is to examine the use of interprofessional learning models to educate a healthcare workforce that meets the needs of older adults by integrating geriatrics with primary care, maximizing patient engagement, and transforming the healthcare system. Specifically, the targeted learners for this curriculum were from a healthcare system in Belize that had no previous specialty training in interprofessional geriatrics care. The 4-day training took place in Belize with an interprofessional group of healthcare professionals that included social work, nursing and medicine. 100 learners participated in the trainings and including participants from social work, nursing and medicine. To evaluate the program, Kirkpatrick’s Training Evaluation Model (Kirkpatrick & Kirkpatrick, 2005) was used to determine if learners were satisfied with the content (reaction), skilled (knowledge & skill) and confident in their abilities to utilize the curriculum (application of knowledge & skills). Analysis showed that learners, irrespective of discipline, were satisfied with the program. All disciplines experienced significant differences in their self-efficacy with working on interdisciplinary teams from pre to post assessments. Specifically, there was an increase in learner’s confidence related to learning to work together cooperatively with other professions and how to communicate effectively with other members of an interprofessional team. Implications for future interprofessional curriculum will be discussed. LONGITUDINAL IMPACT OF AN INTERDISCIPLINARY COURSE ON AGING FOR FIRSTYEAR STUDENTS Matthew Picchiello,1 Nancy Morrow-Howell,2 Susan Stark,2 and Brian Carpenter,2 1. Washington University in St. Louis, University City, Missouri, United States, 2. Washington University in St. Louis, St. Louis, Missouri, United States Undergraduate courses on aging have the potential to counteract negative stereotypes about older adults and to shift students’ academic plans as they learn about agingrelated opportunities. For six years we have taught an interdisciplinary course on aging for first-year undergraduate students. We present longitudinal data on students’ attitudes and academic trajectories after taking the course. Students who took the course (n = 314) and comparable students who were not in the course (n = 353) were surveyed prior to and at the end of their first semester and at the end of each subsequent academic year. At each time point students rated the degree to which aging issues are relevant to their personal and professional lives. Students also reported agingrelated curricular and extracurricular activities they pursued. Multivariate repeated-measures analyses revealed a significant interaction such that personal and professional relevance of aging issues were lower and remained stable for students not in the class, and were higher and increased for students in the class, F(2,226) = 13.18, F(2,226) = 14.94, p’s < .01. However, for course students, relevance returned to baseline levels by the end of their first year and remained constant in subsequent years. Results from chi-square analyses revealed that students in the class reported more engagement in agingrelated courses, χ2(1) = 8.3, research projects, χ2(1) = 90.1, and extracurriculars, χ2(2) = 20.6, p’s < .01. Results suggest that exposing students to information about aging early has the potential to alter academic trajectories, highlighting the importance of early education. LONG-TERM EFFECTS OF ABUSE IN LATER LIFE PERPETRATED BY FAMILY MEMBERS Naomi Meinertz,1 Pi-Ju Liu,2 and Ron Acierno,3 1. Iowa State University, Ames, Iowa, United States, 2. Purdue University, West Lafayette, Indiana, United States, 3. UT Health Sciences Houston, Houston, Texas, United States Abuse in later life could potentially lead to lower levels of social support, especially when perpetrated by family GSA 2020 Annual Scientific Meeting Downloaded from https://academic.oup.com/innovateage/article/4/Supplement_1/444/6037318 by guest on 18 May 2024 ELDER FAMILY FINANCIAL EXPLOITATION: THE COMPLEXITY OF ROLES AND FAMILY CONTEXT Athena Chung Yin Chan,1 and Marlene Stum,2 1. University of Minnesota–Twin Cities, Saint Paul, Minnesota, United States, 2. University of Minnesota, Twin Cities, Saint Paul, Minnesota, United States Despite elder family financial exploitation (EFFE) being recognized as the most prevalent type of elder abuse, little is known about the family context in which it occurs. To-date most EFFE research has focused on understanding the profiles of one victim and one perpetrator in reported cases. Informed by Family Systems Theory, this study offers new insight into the range and complexity of EFFE victim and perpetrator roles, family structures (relationship types across generations) and living arrangements. A mixed-methods dataset from a sample of non-perpetrator/non-victim concerned family members who experienced EFFE (most unreported to authorities) was utilized to map and analyze 23 family system genograms. The findings reflect four overall profiles when organized by the number of victim(s) and perpetrator(s) in each involved family system including: Single victim, single perpetrator (n=7), Single victim, multiple perpetrators (n=12), Two victims, single perpetrator (n=1), and Two victims, multiple perpetrators (n=3). Across the 4 profiles, most primary perpetrators moved in to live with the elder victims. For Single victim, single perpetrator cases, remarried spouses, as well as parent/adult child relationships in nuclear families with 2-3 adult children emerged. For Single victim, multiple perpetrators, up to five family perpetrators from 3 different nuclear families were involved, including adult children, their in-laws, and grandchildren as a common combination of perpetrators. The findings suggest EFFE is more complex than often assumed, involving multiple perpetrators and victims, and family relationship types beyond older parent/adult child. Implications for reframing risk profiles, assessment tools, and family-focused intervention strategies are discussed. Innovation in Aging, 2020, Vol. 4, No. S1 Innovation in Aging, 2020, Vol. 4, No. S1 PREDICTORS OF MOTIVATION TO LEARN AMONG MIDDLE-AGED AND OLDER MEN IN THE UNITED STATES Taka Yamashita,1 Roberto Millar,2 Shalini Sahoo,3 Thomas Smith,4 and Phyllis Cummins,5 1. University of Maryland, Baltimore, Maryland, United States, 2. Graduate Student, Baltimore, Maryland, United States, 3. University of Maryland, Baltimore, Elkridge, Maryland, United States, 4. Northern Illinois University, DeKalb, Illinois, United States, 5. Miami University, Oxford, Ohio, United States Middle-aged and older men are less likely than women to participate in adult education and training (AET) outside of their work. AET is known to provide psychological, social and economic (e.g., job-related skills) benefits throughout the life course. Research has shown that motivation to learn (MtL) is the key to promoting AET. The objective of this study is to identify MtL predictors among middle-aged and older men in the U.S. Nationally representative data (n = 1,450) of men aged 45 years and older were obtained from the 2012/2014 Program for International Assessment of Adult Competencies (PIAAC). Structural equation models were constructed to examine how a latent MtL construct measured by four 5-point Likert-type itemsMtL might be predicted by participant characteristics. Results showed that having a postsecondary degree (vs. high school or less; b = 0.19, p < 0.05), higher literacy skills (0-500 points; b = 0.01, p < 0.05), at least one parent/guardian with a postsecondary degree (vs. those without; b = 0.08, p < 0.05) and better self-rated health (b = 0.14, p < 0.05) were associated with greater MtL. Additionally, Black (b = 0.22, p < 0.05) and Hispanic (b = 0.19, p < 0.05) men showed greater MtL than White men. Overall, socioeconomic status indicators and race/ethnicity were linked to MtL. Given the known challenges involved in middle-aged and older men’s participationin AET (e.g., low and short-term participation), enhancing MtL may have long-term implications. Theoretical explanations and possible policy implications are evaluated. GSA 2020 Annual Scientific Meeting RETIREMENT PREPAREDNESS: HOW IMPORTANT IS BEING FINANCIALLY LITERATE? Mengya Wang,1 and Suzanne Bartholomae,2 1. Iowa State University, ames, Iowa, United States, 2. Iowa State University, Ames, Iowa, United States Financial security in retirement is a major concern for many Americans. Numerous studies document that Americans are not prepared for retirement, with financial illiteracy cited as one reason Americans fail to plan. Employing data from the 2018 National Financial Capability Study (N=27,091), this study investigates actual financial literacy (AFL) and perceived financial literacy (PFL) and how combinations of this measure influences retirement planning, and varies based on years from retirement. This study found relatively low financial literacy and retirement preparedness levels among the US sample, even for those pre-retirees ages 55 to 64. Individually, PFL and AFL increased as one approached retirement. When combined, adults nearing retirement (55 to 64) comprised the greatest proportion of the high AFL and high PFL (29.9%) group compared to adults 20 years or more from retirement (18-44) who largely made up the low AFL and PFL (48%) group. Based on a logistic regression, adults closest to retirement (ages 55 to 64) are more likely to be planning compared to the other groups, as are adults who were financially confident, risk takers, highly educated, males, and white. Compared to adults with high AFL and high PFL, adults with low AFL and low PFL, or a combination (low PFL and high AFL, high PFL and low AFL) have lower odds of preparing for retirement. Both PFL and AFL influences retirement planning, and PFL may be as important as AFL. Our highlight the importance of policies and programs to support Americans with retirement planning. SEASON’S CHEATINGS: BEWARE OF HOLIDAY SCAMS Lona Choi-Allum, and Alicia Williams, AARP, Washington, District of Columbia, United States ‘Tis the season…to be on the lookout for possible scams and fraud. It’s during this time of year that individuals are more focused on the spirit of the holidays and less focused on what may be happening with their pocketbook. AARP conducted a survey of 2,842 U.S. adults ages 18 and older to understand people’s awareness of and experience with a variety of scams that are common around the holidays. The study explored experiences with purchasing gift cards, shipping/receiving packages, and charitable giving. In addition, the survey tested the knowledge of adults about several specific scams with a ‘quiz’ of five true or false statements. Results showed that one in six (17%) U.S. adults failed the quiz. When making gift card purchases, one in five U.S. adults have given and/or received a gift card that had no funds on it. And only about half of U.S. adults conduct research before making a monetary donation to charitable causes or organizations. Of those who do check out a charity first, over half (54%) did not make a donation based on what they found on charity rating sites. Also, U.S. adults say that packages are left outside of their home without requiring a signature. Half (50%) of U.S. adults say they never require a signature Downloaded from https://academic.oup.com/innovateage/article/4/Supplement_1/444/6037318 by guest on 18 May 2024 members who are charged with protecting the older adult in their care. Using both waves of the National Elder Mistreatment longitudinal data (wave one collected in 2008 and wave two in 2015; N=774), long-term effects of abuse (i.e., physical, emotional, sexual, and financial) on levels of social support, physical health, and clinical depressive symptoms for respondents at or above the age of 60 years were analyzed. A multivariate analysis of variance showed that respondents abused at wave one (n=261) by a family member (B=-0.55, p≤0.001), a spouse or ex-partner (B=-0.349, p=0.02), or a non-relative or stranger (B=-0.301, p=0.026) had lower levels of social support eight years later at wave two. Those abused by a family member at wave one also experienced higher levels of depressive symptoms at wave two (B=-0.187, p=0.01). Perpetrator type did not predict general health at wave two. These results emphasize the long-term impact of abuse on the lives of older adults and highlight the importance trusted relationships, such as with family members, have on older adult health and wellbeing. 445 

W3159487329.txt

Published for SISSA by Springer Received: February 9, Revised: March 30, Accepted: March 30, Published: April 26, 2021 2021 2021 2021 SU(5) GUTs with A4 modular symmetry a College of Information Science and Engineering, Ocean University of China, Qingdao 266100, China b Peng Huanwu Center for Fundamental Theory, Hefei, Anhui 230026, China c Interdisciplinary Center for Theoretical Study and Department of Modern Physics, University of Science and Technology of China, Hefei, Anhui 230026, China d Physics and Astronomy, University of Southampton, Southampton, SO17 1BJ, U.K. E-mail: pche@mail.ustc.edu.cn, dinggj@ustc.edu.cn, king@soton.ac.uk Abstract: We combine SU(5) Grand Unified Theories (GUTs) with A4 modular symmetry and present a comprehensive analysis of the resulting quark and lepton mass matrices for all the simplest cases. Classifying the models according to the representation assignments of the matter fields under A4 , we find that there are seven types of SU(5) models with A4 modular symmetry. We present 53 benchmark models with the fewest free parameters. The parameter space of each model is scanned to optimize the agreement between predictions and experimental data, and predictions for the masses and mixing parameters of quarks and leptons are given at the best fitting points. The best fit predictions for the leptonic CP violating Dirac phase, the lightest neutrino mass and the neutrinoless double beta decay parameter when displayed graphically are observed to cover a wide range of possible values, but are clustered around particular regions, allowing future neutrino experiments to discriminate between the different types of models. Keywords: Discrete Symmetries, GUT, Neutrino Physics, Quark Masses and SM Parameters ArXiv ePrint: 2101.12724 Open Access, c The Authors. Article funded by SCOAP3 . https://doi.org/10.1007/JHEP04(2021)239 JHEP04(2021)239 Peng Chen,a Gui-Jun Dingb,c and Stephen F. Kingd Contents 1 Introduction 1 2 Modular symmetry and modular forms of level N = 3 4 7 8 9 11 12 4 Benchmark models 4.1 Type-I 4.2 Type-II 4.3 Type-V 4.4 Type-VII 13 14 25 26 39 5 Numerical results 42 6 Conclusion 46 A Results of the best fit for the benchmark models 48 1 Introduction The standard model (SM) of electroweak interactions based on SU(2)L ×U(1)Y is sixty years old this year [1], although the inclusion of the Higgs mechanism and QCD required a further decade of work as has been well documented elsewhere. It is by now well established and has successfully passed many precision tests at various energy levels. After the discovery of the Higgs boson at the LHC, all particles predicted by the three family SM have been observed. Although there is no concrete experimental evidence for new physics beyond the SM, apart from the neutrino masses and mixing, most physicists believe that the SM should be a low energy effective theory rather than a fundamental theory. One essential question of the SM is how to understand the hierarchical mass spectrum and flavor mixing patterns of quarks and leptons, including neutrinos whose masses lie beyond the SM. The masses of quarks and leptons span about 13 orders of magnitude. The Planck collaboration gives P i mi < 0.120 eV which implies the lightest neutrino mass is smaller than 0.04 eV [2], while the top quark mass is around 173 GeV. The three quark mixing angles are hierarchical and q the largest one is the Cabibbo angle θ12 ' 13.09◦ , and the CP phase of the quark sector is q l ' 33.44◦ and δCP ' 68.53◦ [3]. In contrast to the quark sector, the solar mixing angle θ12 –1– JHEP04(2021)239 3 General form of fermion mass matrices 3.1 Majorana mass matrix of the right-handed neutrinos 3.2 Dirac neutrino Yukawa coupling 3.3 The charged lepton and down quark sectors 3.4 The up quark sector –2– JHEP04(2021)239 l ' 49.2◦ are large, and the reactor mixing angle is small the atmospheric mixing angle θ23 l ' 8.57◦ , while the CP violation of the lepton sector has not been confirmed yet [4]. with θ13 The quark and charged lepton masses are described by different interaction strengths with the Higgs doublets within the SM, but their values cannot be predicted. Understanding the flavor structure of the SM from the first principles is one of the greatest challenges in particle physics. Much effort has been devoted to addressing the flavor puzzle, and symmetry has been an important guiding principle. It is found that the non-Abelian discrete flavor symmetry is particularly suitable to explain the large lepton mixing angles l and θ l [5]. In conventional flavor symmetry models, a complicated vacuum alignment θ12 23 is frequently required, since flavons are generally necessary and their vacuum expectation values (VEVs) should be oriented along certain directions in flavor space. Recently modular invariance has been suggested to play the role of flavor symmetry, especially in the neutrino sector [6], an approach which is inspired by superstring theory with compactified extra dimensions. The finite discrete flavor symmetry groups arise as the quotient group of modular group SL(2, Z) over the principal congruence subgroups. The quark and lepton fields transform nontrivially under the finite modular groups and are assigned to various modular weights, thus modular invariance requires the Yukawa couplings are modular forms which are holomorphic functions of the complex modulus τ . The flavon fields other than the modulus might not be needed and the flavor symmetry can be entirely broken by the vacuum expectation value (VEV) of the modulus τ . Moreover, all higher dimensional operators in the superpotential are completely fixed by the modular invariance. The modular form of level N and integral weight k can be arranged into some modular multiplets of the homogeneous finite modular group Γ0N ≡ Γ/Γ(N ) [7], and they can be organized into modular multiplets of the inhomogeneous finite modular group ΓN ≡ Γ/Γ(N ) if k is an even number [6]. The inhomogeneous finite modular group ΓN of lower levels N = 2 [8–11], N = 3 [6, 8, 9, 12–37], N = 4 [25, 38–45], N = 5 [43, 46, 47] and N = 7 [48] have been considered and a large number of models have been constructed. All the modular forms of integral weights can be generated from the tensor products of weight one modular forms and the odd weight modular forms are in the representations with ρr (S 2 ) = −1. The homogeneous finite modular groups Γ0N provide richer structure of modular forms for flavor model building, and the small groups Γ03 ∼ = T 0 [7, 49], Γ04 ∼ = S40 [50, 51] and Γ05 ∼ = A05 [52] have been studied. If the modular weight k of the operator is not an integer, (cτ +d)k is not the automorphy factor anymore and some multiplier is needed, consequently the modular group should be extended to its metaplectic covering [53]. The framework of modular invariance have been extended to include the modular forms of rational weights k/2 [53] and k/5 [54]. The generalized CP symmetry can be consistently imposed in the context of modular symmetry, the modulus is determined to transform as τ → −τ ∗ under the action of CP [55–57]. The CP transformation matrix is completely fixed by the consistency condition up to an overall phase, and it is exactly the canonical CP transformation in the symmetric basis [55]. The more fundamental theory such as string theory sometimes requires several compact space with more than one modulus parametrizing its shape. The modular invariance approach has been extended to incorporate several factorizable [40] and non-factorizable moduli [58]. –3– JHEP04(2021)239 Grand unified theories (GUTs) are amongst the most well motivated theories beyond the SM, realising the elegant aspiration to unify the three gauge interactions of the SM into a simple gauge group [59]. The fermion representations of the SM are unified into a smaller number of multiplets under the GUT gauge group, with quarks and leptons being assigned to the same GUT multiplet, thereby providing an explanation for charge quantisation. Imposing a family symmetry together with GUTs can help to address the problem of quark and lepton mass and mixing hierarchies [60]. Among many different GUT scenarios, SU(5) [59] is the minimal choice, being the smallest simple group which can accommodate the gauge symmetry of the SM. Although neutrino masses are not predicted by SU(5), they can easily be accommodated as singlet representations of the GUT group. Among the many choices of family symmetry, A4 is the minimal choice which admits triplet representations [61]. Combining A4 family symmetry with SU(5) GUTs [62] also requires vacuum alignment of the flavons in order to break the A4 , and so there is a strong motivation for introducing modular symmetry in such frameworks. Indeed, modular symmetry in the context of SU(5) GUTs was first studied in an (Γ3 ' A4 ) × SU(5) model in [14]. Other modular GUT models were subsequently constructed based on (Γ2 ' S3 ) × SU(5) [10, 63], and (Γ4 ' S4 ) × SU(5) [64]. In this paper we shall perform a comprehensive study of the Γ3 ' A4 modular symmetry in the framework of supersymmetric (SUSY) SU(5) GUTs. It is known that the fifteen matter fields in each generation are embedded into two chiral supermultiplets 5 and 10. The SU(5) gauge symmetry is spontaneously broken down to the SM gauge group SU(3)c × SU(2) × U(1)Y by the VEV of the Higgs H24 in the adjoint representation of SU(5). In the minimal SU(5) model, two Higgs multiplets H5 and H5 in the fundamental representation 5 and antifundamental representation 5 of SU(5) further break the SM gauge symmetry into SU(3)c × U(1)EM . The charged leptons and down-type quarks are in the same GUT multiplets, the down type quark mass matrix is the transpose of the charged lepton mass matrix in the minimal SU(5) such that the masses of the charged leptons and down quarks would be identical. In order to account the different masses of down quarks and charged leptons, additional Higgs multiplet H45 is introduced in our models [65]. The neutrinos are massless in the minimal SU(5) GUT because of the absence of right-handed neutrinos. In the present work, we extend the matter contents by including right-handed neutrinos which are SU(5) singlets, and the neutrino masses are generated by the type-I seesaw mechanism [66–70]. The most minimal version of the seesaw mechanism involves two additional right-handed neutrinos [71, 72], consequently both versions of seesaw models with two and three right-handed neutrinos are considered. The A4 group has three singlet representations 1, 10 , 100 and a triplet representation 3. We assume the three Higgs fields are modular invariants and their modular weights are vanishing, and the three generations of matter fields are assigned to transform as singlets or triplet of A4 . The purpose of this work is to find phenomenologically viable SU(5) GUT models based on A4 modular symmetry with less parameters. We classify all possible A4 modular SU(5) GUT models into seven different types according to transformation properties of the matter superfields under A4 . Intensive numerical analysis is performed for each model and we have optimized the free parameters of the models in order to match the experimental data. 2 Modular symmetry and modular forms of level N = 3 The modular group SL(2, Z) consists of matrices with integer entries and determinant 1, it acts on the upper half plane as fractional linear(Mobius) transformations: aτ + b τ→ 7 γτ = , cτ + d a b c d ! ∈ SL(2, Z), Im(τ ) > 0 . (2.1) It is easily verified that the kernel of this action is the center C = {I, −I} where I denotes the two dimensional unit matrix. Therefore the group of fractional linear transformations is isomorphic to the projective special linear group PSL(2, Z) ∼ = SL(2, Z)/C = Γ which is the quotient of SL(2, Z) by its center C. Although the group PSL(2, Z) is of infinite order, it can be generated by two transformations S and T , S= 0 −1 T = 1 0 : 1 τ →− , τ : τ → τ + 1. ! 1 0 ! 1 1 (2.2) Taking the quotient of PSL(2, Z) over the principal congruence subgroup of level N , we can obtain the (inhomogeneous) finite modular group ΓN ∼ = Γ/Γ(N ) with ( Γ(N ) = a b c d ! ! ∈ SL(2, Z), a b (mod N ) = c d !) 1 0 0 1 , (2.3) and Γ(N ) = Γ(N ) for N > 2 and Γ(N ) = Γ(N )/{I, −I} for N = 1, 2. Obviously T N ∈ Γ(N ), consequently the finite modular group ΓN can be generated by the generators S and T satisfying S 2 = (ST )3 = T N = 1 . (2.4) Additional relations are needed to render the group finite make for N > 5 [48]. In the present we are interested in the minimal finite modular group Γ3 = Γ/Γ(3) ∼ = A4 which –4– JHEP04(2021)239 This paper is organized as follows. In section 2, we briefly review modular symmetry and modular forms, and modular forms of level 3 are listed. In section 3, we present the most general form of the A4 modular quark and lepton mass matrices in SU(5), and all possible A4 representation assignments of matter fields are considered. In section 4, according to the representation assignments of matter fields, we find that there are seven different types of SU(5) GUT models with A4 modular symmetry, and some benchmark models are presented. We don’t consider the cases in which no matter fields are assigned to triplet of A4 since generally more free parameters would be involved. In section 5, we show the results of a scan of the parameter space for each benchmark model, we search for the minimum of the χ2 function to optimize the agreement between predictions and experimental data. Finally we draw the conclusions in section 6. Appendix A gives the best fit values of the input parameters and the predictions for the masses, mixing angles and CP violating phases of quarks and leptons. can be seen as the symmetry group of the tetrahedron. There are 4 inequivalent irreducible representations of A4 : three singlets 1, 10 , 100 and a triplet 3. In the three singlet representations, the generators S and T are 1 : S = 1, T = 1, 10 : S = 1, T = ω, 00 1 : S = 1, T = ω2 , (2.5) −1 2 2 1  S =  2 −1 2  , 3 2 2 −1   1  T = 0 0  0 ω 0 0  0. ω2  (2.6) The decompositions of the direct product of A4 representations are 10 ⊗ 10 = 100 , 10 ⊗ 100 = 1, 100 ⊗ 100 = 10 , 3 ⊗ 3 = 1 ⊕ 10 ⊕ 100 ⊕ 3 ⊕ 3 . (2.7) Given two triplets α = (α1 , α2 , α3 ) and β = (β1 , β2 , β3 ), the irreducible representations obtained from their product are: 1 = α1 β1 + α2 β3 + α3 β2 , 10 = α3 β3 + α1 β2 + α2 β1 , 100 = α2 β2 + α1 β3 + α3 β1 , 3S = (2α1 β1 − α2 β3 − α3 β2 , 2α3 β3 − α1 β2 − α2 β1 , 2α2 β2 − α1 β3 − α3 β1 ) , 3A = (α2 β3 − α3 β2 , α1 β2 − α2 β1 , α3 β1 − α1 β3 ) , (2.8) where 3S(A) denotes the symmetric (antisymmetric) combination. The modular forms of level N and even weight k span a linear space of finite dimension, and they can be organized (k) into some modular multiplets Yr (τ ) transforming in the irreducible representation r of ΓN up to the automorphy factor, (k) Yr (γτ ) = (cτ + d) k (k) ρr (γ)Yr (τ ), ∀γ = a b c d ! ∈ Γ. (2.9) The modular forms of level 3 has been constructed [6], and it turns out that there are only three linearly independent modular forms Y1,2,3 (τ ) of weight 2 and level 3, i η 0 (τ /3) η 0 ((τ + 1)/3) η 0 ((τ + 2)/3) 27η 0 (3τ ) + + − , 2π η(τ /3) η((τ + 1)/3) η((τ + 2)/3) η(3τ )   0 −i η 0 (τ /3) η 0 ((τ + 2)/3) 2 η ((τ + 1)/3) Y2 (τ ) = +ω +ω , π η(τ /3) η((τ + 1)/3) η((τ + 2)/3)   −i η 0 (τ /3) η 0 ((τ + 1)/3) η 0 ((τ + 2)/3) Y3 (τ ) = +ω + ω2 , π η(τ /3) η((τ + 1)/3) η((τ + 2)/3) Y1 (τ ) =   –5– (2.10) JHEP04(2021)239 where ω = e2πi/3 refers to a cubic root of unity. We shall denote 1 ≡ 10 , 10 ≡ 11 and 100 ≡ 12 when constructing models in the following. The triplet representation 3 in the bassi where T is diagonal is given by where η(τ ) is the famous Dedekind eta-function, η(τ ) = q ∞ Y 1/24 (1 − q n ), q = e2πiτ . (2.11) n=1 Thus the q-expansion of Y1,2,3 (τ ) reads Y1 (τ ) = 1 + 12q + 36q 2 + 12q 3 + 84q 4 + 72q 5 + . . . , Y2 (τ ) = −18q 2/3 (1 + 2q + 5q 2 + 4q 3 + 8q 4 + . . . ) . (2.12) (2) The above three modular forms can be arranged into a triplet Y3 = (Y1 , Y2 , Y3 )T which transforms as a three-dimensional irreducible representation of A4 up to the automorphy factor: (2) S (2) (2) Y3 (τ ) − → Y3 (−1/τ ) = τ 2 ρ3 (S)Y3 (τ ), T (2) (2) (2) Y3 (τ ) − → Y3 (τ + 1) = ρ3 (T )Y3 (τ ) , (2.13) where ρ3 (S) and ρ3 (T ) are the representation matrices of S and T respectively given in eq. (2.6). The weight 2k modular forms of level 3 are homogeneous polynomials Yi1 Yi2 . . . Yik of degree k in Yi (τ ). There are five linearly independent weight four modular forms and they decompose as 3 ⊕ 1 ⊕ 10 under A4 ,  2  Y1 − Y2 Y3 1 (2) (2) (4)   Y3 = (Y3 Y3 )3 = Y32 − Y1 Y2  , 2 Y22 − Y1 Y3 (4) Y1 (2) (2) (2) (2) = (Y3 Y3 )1 = Y12 + 2Y2 Y3 , (4) Y10 = (Y3 Y3 )10 = Y32 + 2Y1 Y2 . (2.14) The tensor products of weight 2 and weight 4 modular forms give rise to weight 6 modular forms, (6) Y1 (6) (2) (4) = (Y3 Y3 )1 = Y13 + Y23 + Y33 − 3Y1 Y2 Y3 , (2) (4) Y3I = Y3 Y1 (6) (2) (4) Y3II = Y3 Y10     Y1   2 = (Y1 + 2Y2 Y3 ) Y2  , Y3 Y3   2 = (Y3 + 2Y1 Y2 ) Y1  . Y2 –6– (2.15) JHEP04(2021)239 Y2 (τ ) = −6q 1/3 (1 + 7q + 8q 2 + 18q 3 + 14q 4 + . . . ) , weight k representation r 2 3 4 1, 10 , 3 6 1, 3, 3 8 1, 10 , 100 , 3, 3 (k) Table 1. The transformation properties of the weight k and the level 3 modular forms Yr under A4 . (8) (2) (6) (8) (2) (6) (8) (2) (6) Y1 = (Y3 Y3I )1 = (Y12 + 2Y2 Y3 )2 , Y10 = (Y3 Y3I )10 = (Y12 + 2Y2 Y3 )(Y32 + 2Y1 Y2 ) , Y100 = (Y3 Y3II )100 = (Y32 + 2Y1 Y2 )2 ,  (8) (2) (6) Y3I = Y3 Y1  Y1   = (Y13 + Y23 + Y33 − 3Y1 Y2 Y3 ) Y2  , Y3  2  Y2 − Y1 Y3 (8) (2) (6)   Y3II = (Y3 Y3II )3A = (Y32 + 2Y1 Y2 ) Y12 − Y2 Y3  . (2.16) Y32 − Y1 Y2 Notice that modular forms in all irreducible representations of A4 appears at weight 8, both (6) (8) (2) Y3I (τ ) and Y3I (τ ) are proportional to Y3 (τ ). We summarize the even weight modular forms of level 3 and their transformation under A4 in table 1. 3 General form of fermion mass matrices We formulate our models in the framework of supersymmetric SU(5) GUT. The neutrino masses are generated by the type-I seesaw mechanism [66–70], consequently three (or two) right-handed neutrinos N are introduced and they are SU(5) singlets. Thus all left-handed quark and lepton superfields within each family are embedded into three SU(5) multiplets 1, 5 and 10 with N = νc ,  c dr  c  dg  c F =  db   e −ν     ,    0 ucb −ucg   −ucb 0 ucr  c c T = 0  ug −ur  u u u  r g b dr dg db   −ur −dr  −ug −dg   −ub −db  ,  0 ec  −ec 0 (3.1) where the superscript c denotes charge conjugation of the right-handed superfields. The weight and transformation of the above matter superfields under A4 are denoted as kψ and ρψ respectively with ψ = N, F , T . Moreover, the SU(5) Higgs fields H5 and H5 , H45 is –7– JHEP04(2021)239 The weight 8 modular forms which can be arranged into two triplets and three singlets of A4 considered to account for the mass differences of down quarks and charged leptons. All the three Higgs fields are assumed to be invariant under A4 and their modular weights are zero. Each of these GUT Higgs representations contains an SU(2)L Higgs doublet, the low energy doublet Hu in the minimal supersymmetric standard model arises from H5 , and Hd originates from a linear combination of H5 and H45 . The three generations of F , T and N can transform as either a triplet 3 or three singlets under A4 modular symmetry. The most general superpotential for quark and lepton masses is of the following form, 0 W = N N fM (Y ) + N F H5 fN (Y ) + F T H5 fD (Y ) + F T H45 fD (Y ) + T T H5 fU (Y ) , (3.2) 3.1 Majorana mass matrix of the right-handed neutrinos The first term N N fM (Y ) in eq. (3.2) gives rise to Majorana mass matrix of the righthanded neutrinos, and the modular function fM (Y ) is completely fixed by the weight and representation of N . • ρN = 3 Using the contraction rules in eq. (2.8), we can read off the mass matrix of the righthanded neutrinos for different values of N ’s weight kN , (0) k N = 0 : MN = Λ S1 , (2) k N = 1 : MN = Λ S3 , (4) (4) (6) Λ1 S3I (8) Λ1 S3I (6) Λ2 S3II (8) Λ2 S3II (3.3) (4) k N = 2 : MN = Λ 1 S3 + Λ 2 S 1 + Λ 3 S 1 0 , k N = 3 : MN = k N = 4 : MN = + + + + (6) Λ 3 S1 , (8) Λ 3 S1 + (8) (8) Λ4 S10 + Λ5 S100 , where Λi are the characteristic scale of flavour dynamics. For simplicity we have defined 1 0 0 (k)   = Y1 (τ ) 0 0 1 , 0 1 0  (k) S1  0 0 1 (k)   = Y10 (τ ) 0 1 0 , 1 0 0  (k) S 10  0 1 0 (k)   = Y100 (τ ) 1 0 0 , 0 0 1 (3.4)  (k) S100  and  (k) (k) (k)  2Y (τ ) −Y3,3 (τ ) −Y3,2 (τ )  3,1  (k) (k) (k) (k)  S3 (τ ) =  −Y3,3 (τ ) 2Y3,2 (τ ) −Y3,1 (τ ) , (k) (k) (k) −Y3,2 (τ ) −Y3,1 (τ ) 2Y3,3 (τ ) (0) where we denote Y1 (τ ) = 1 and 3 stands for 3, 3I or 3II. –8– (3.5) JHEP04(2021)239 0 (Y ) and f (Y ) are functions of modular forms, and they where fM (Y ), fN (Y ), fD (Y ), fD U can be determined by the requirement that each term of W is modular invariant with total vanishing modular weight. Because both H5 and H45 are assumed to be A4 invariant 0 (Y ) singlet and their modular weights are vanishing, the modular functions fD (Y ) and fD are of the same form. In the following, we shall present the most general form of the mass matrices of quarks and leptons in SU(5) models with A4 modular symmetry. We shall consider modular forms up to weight 8 in the following, and extension to higher weight modular forms is straightforward. • ρN = 1, 10 , 100 In this case, fM (Y ) has to be modular form transforming as singlet under A4 . It is known that the mass matrix MN of right-handed neutrinos is a symmetric matrix. For the modular weight kNi +kNj ≤ 8, the possible nonzero elements depend on their weights and A4 kronecker products M kNi + kNj = 0 : (MN )ij = αij Λ for ρNi ⊗ ρNj = 1 , kNi + kNj = 4 : (MN )ij = for ρNi ⊗ ρNj = 1 ,  M (4) αij ΛY10 (τ ) for ρNi ⊗ ρNj = 100 , (6) M kNi + kNj = 6 : (MN )ij = αij ΛY1 (τ ) for ρNi ⊗ ρNj = 1 , kNi + kNj = 8 : (MN )ij =  M ΛY (8) (τ )  αij  1    (8) αij ΛY100 (τ )      M (8) M αij ΛY10 (τ ) for ρNi ⊗ ρNj = 1 , for ρNi ⊗ ρNj = 10 , (3.6) for ρNi ⊗ ρNj = 100 , M are coupling constants. where αij 3.2 Dirac neutrino Yukawa coupling The Dirac neutrino Yukawa coupling arises from the term N F H5 fN (Y ) in eq. (3.2). In the following, we report the Dirac neutrino Yukawa coupling for different weight and representation assignments of N and F . • ρN = ρF = 3 For different values of kN and kF , we can read out the neutrino Yukawa couplings Y5ν as follows. (0) kN + kF = 0 : Y5ν = S1 , (2) (2) (4) (4) (4) (6) (6) (6) (6) (6) (8) (8) (8) (8) (8) kN + kF = 2 : Y5ν = S3 + A3 , (4) kN + kF = 4 : Y5ν = S3 + A3 + S1 + S10 , kN + kF = 6 : Y5ν = S3I + A3I + S3II + A3II + S1 , (8) (8) kN + kF = 8 : Y5ν = S3I + A3I + S3II + A3II + S1 + S10 + S100 , (3.7) where we have omitted the coupling coefficients of each independent term and  (k) 0 Y3,3 (τ )  (k) (k)  A3 (τ ) = −Y3,3 (τ ) 0 (k) (k) Y3,2 (τ ) −Y3,1 (τ ) (k)  − Y3,2 (τ )  (k) Y3,1 (τ )  . 0 (3.8) • ρN = 3, ρF = 1, 10 , 100 In this case, the modular function f N (Y ) should be a modular form in the representation 3 of A4 . For notational simplicity, we denote 10,1,2 = 1, 10 , 100 and (ρF 1 , ρF 2 , ρF 3 ) = –9– JHEP04(2021)239  M ΛY (4) (τ )  αij 1 a a a (1 F 1 , 1 F 2 , 1 F 3 ) with aF j = 0, 1, 2. Then the (ij) element of the Dirac neutrino Yukawa coupling matrix is given by, (kN +kF ) kN + kF j = 2, 4 : (Y5ν )ij = ανj Y3,hi+a kN + kF j = 6, 8 : (Y5ν )ij j Fj i (kN +kF ) = ανj Y3I,hi+a j Fj i , + (kN +kF ) αν0 j Y3II,hi+a j i , (3.9) Fj kN +kF Y5ν = CaF 1 1 kN +kF , C aF 2 2 kN +kF , C aF 3 (3.10) , 3 with kN +kF C aF j j  (k +k )  N Fj Y3,h1+aF i (τ )   j  (kN +k )  Fj   = Y3,h2+a i (τ ) .   Fj  (k +k )  N   Fj Y3,h3+a Fj (3.11) (τ ) i To be more specific, we have kN +kF kN +kF j = 2, 4 : CaF kN +kF kN +kF j = 6, 8 : CaF j j j j =   (kN +kF ) (kN +kF ) (kN +kF ) T  j j j   Y , Y , Y  3,1 3,3 3,2        (kN +k ) (kN +k ) (kN +k ) T Y Fj ,Y Fj ,Y Fj 3,3 3,2 3,1        (kN +kF ) (kN +kF ) (kN +kF ) T   j j j   Y3,2 , Y3,1 , Y3,3    (kN +kF ) (kN +kF ) (kN +kF ) T   j j j  Y , Y , Y  3I,1 3I,3 3I,2        (kN +kF ) (kN +kF ) (kN +kF ) T   j j j   + Y3II,1 , Y3II,3 , Y3II,2        (k +k ) (k +k ) (k +k ) T   N N N  Fj Fj Fj   , Y3I,2 , Y3I,1  Y3I,3 =  (k +k ) (k +k ) (k +k ) T  N N N F F F    + Y3II,3 j , Y3II,2 j , Y3II,1 j        (k +k ) (k +k ) (k +k ) T   N N N Fj Fj Fj    Y , Y , Y  3I,2 3I,1 3I,3       (kN +kF ) (kN +kF ) (kN +kF ) T   j j j  ,Y ,Y + Y 3II,2 3II,1 3II,3 for aF j = 0 , for aF j = 1 , for aF j = 2 , for aF j = 0 , for aF j = 1 , for aF j = 2 , where we have omitted the overall coefficient associated with each independent contraction for every column. – 10 – JHEP04(2021)239 with h1i = h4i = 1, h2i = h5i = 3 and h0i = h3i = 2. Here i and j are the generation indices of N and F respectively. The neutrino Yukawa couplings can be compactly written as   • ρN = 1, 10 , 100 , ρF = 3 Similar to previous case, we denote (ρN1 , ρN2 , ρN3 ) = (1aN1 , 1aN2 , 1aN3 ) with aNi = 0, 1, 2 and the neutrino Yukawa couplings read as (kN +k ) kNi + kF = 2, 4 : i F (Y5ν )ij = ανi Y3,hj+a , N i kNi + kF = 6, 8 : (Y5ν )ij i = (kNi +kF ) ανi Y3I,hj+a Ni i (kN +k ) i F + αν0 i Y3II,hj+a , N i i (3.12)  k +k N F kN +kF Y5ν = CaN11 3.3 , CaN22  kN +kF T , CaN33 (3.13) . The charged lepton and down quark sectors The masses of the charged lepton and down type quarks are described by the terms F T H5 fd (Y ) and F T H45 fd0 (Y ) in eq. (3.2). Modular invariance strongly constrains the 0 (Y ). form of the modular functions fD (Y ) and fD • ρF = ρT = 3 The Yukawa coupling with the Higgs H5 is determined by the modular weight of matter fields. (0) kF + kT = 0 : Y5d = S1 , (2) (2) (4) (4) (4) (6) (6) (6) (6) (6) (8) (8) (8) (8) (8) kF + kT = 2 : Y5d = S3 + A3 , (4) kF + kT = 4 : Y5d = S3 + A3 + S1 + S10 , kF + kT = 6 : Y5d = S3I + A3I + S3II + A3II + S1 , (8) (8) kF + kT = 8 : Y5d = S3I + A3I + S3II + A3II + S1 + S10 + S100 , (3.14) where the dimensionless coefficient in front of each term has been omitted. Analogously d with H the Yukawa coupling Y45 45 can be obtained, and it is found to be of the same d form as Y5 because both H5 and H45 are assumed to be A4 singlets with vanishing modular weight. • ρF = 3, ρT = 1, 10 , 100 The charged lepton and down quark Yukawa couplings depend on the A4 representation of the ten-dimensional matter fields T and their weights. We can read out (k +kT ) j F kF + kTj = 2, 4 : (Y5d )ij = αdj Y3,hi+a , T i j kF + kTj = 6, 8 : (Y5d )ij (kF +kTj ) (k +kT ) j F = αdj Y3I,hi+aT i + αd0 j Y3II,hi+a , T i j j (3.15) where αdj and αd0 j are coupling constants, and i and j stand for the flavor indices of F d is of the same form as Y d . and T respectively. Note Y45 5 – 11 – JHEP04(2021)239 where i and j are the flavor indices of N and F respectively, ανi and αν0 i are free coupling constants. We can also write • ρF = 1, 10 , 100 , ρT = 3 For this assignment, the (ij) element of the Yukawa coupling matrix is found to be (kF +kT ) i kF i + kT = 2, 4 : (Y5d )ij = αdi Y3,hj+a Fi i , (kF +kT ) i kF i + kT = 6, 8 : (Y5d )ij = αdi Y3I,hj+a Fi (kF +kT ) 0 i i + αdi Y3II,hj+a Fi i. (3.16) The Georgi-Jarlskog factor “−3” can account for the mass differences of charged leptons and down quarks [65]. 3.4 The up quark sector The up quark mass matrix is symmetric in the SU(5) GUT theory and it is predicted to be of the same form as the right-handed neutrino mass matrix MN in the current context of A4 modular symmetry. • ρT = 3 We can read out the up quark Yukawa coupling matrix for different values of modular weight kT as follows, (0) kT = 0 : Y5u = S1 , (2) kT = 1 : Y5u = S3 , (4) (4) (4) (6) (6) (6) (8) (8) (8) kT = 2 : Y5u = S3 + S1 + S10 , kT = 3 : Y5u = S3I + S3II + S1 , (8) (8) kT = 4 : Y5u = S3I + S3II + S1 + S10 + S100 . (3.18) • ρT = 1, 10 , 100 For the modular weight kTi + kTj ≤ 8, the possible non-vanishing elements of Y5u are u kTi + kTj = 0 : (Y5u )ij = αij for ρTi ⊗ ρTj = 1 , kTi + kTj = 4 : (Y5u )ij =  u Y (4) (τ )  αij 1 for ρTi ⊗ ρTj = 1 ,  u (4) αij Y10 (τ ) for ρTi ⊗ ρTj = 100 , (6) u kTi + kTj = 6 : (Y5u )ij = αij Y1 (τ ) for ρTi ⊗ ρTj = 1 , kTi + kTj = 8 : (Y5u )ij =  u Y (8) (τ )  αij  1    (8) αij Y100 (τ )      u (8) u αij Y10 (τ ) – 12 – for ρTi ⊗ ρTj = 1 , for ρTi ⊗ ρTj = 10 , for ρTi ⊗ ρTj = 100 . (3.19) JHEP04(2021)239 The electroweak Higgs field Hd is a linear combination of the doublet components of the Higgs fields H5 and H45 , and the mixing angle can be absorbed into the coupling constants. Hence the charged lepton and down quark mass matrices are of the following form: d T d Me = (Y5d − 3Y45 ) vd , Md = (Y5d + Y45 )vd . (3.17) 4 Benchmark models We can classify all the possible A4 modular SU(5) models according to the transformation properties of the matter fields N , F and T under A4 . We don’t consider the cases in which all matter fields are assigned to A4 singlets, since the Yukawa superpotentials would be less constrained by modular symmetry and generally more free parameters would be involved to produce the observed mass and mixing patterns of quarks and leptons. If three right-handed neutrinos are introduced in the seesaw mechanism, we have the following five types of models: N ∼ 3, Type-II : 0 N1,2,3 ∼ 1(10 , 100 ) , Type-IV : N ∼ 3, N1,2,3 ∼ 1(10 , 100 ) , Type-V : 00 F 1,2,3 ∼ 1(1 , 1 ) , N ∼ 3, Type-III : T1,2,3 ∼ 1(10 , 100 ) , F ∼ 3, (4.1) T ∼ 3, (4.2) F ∼ 3, T ∼ 3, (4.3) F ∼ 3, T ∼ 3, (4.4) T1,2,3 ∼ 1(10 , 100 ) . F ∼ 3, (4.5) As shown in ref. [20], there are ten possibilities for the singlet assignment of the three generations of matter fields, C1 : {1, 1, 1} , C2 : {10 , 10 , 10 } , C3 : {100 , 100 , 100 } , C4 : {1, 1, 10 } , C5 : {1, 1, 100 } , C6 : {10 , 10 , 1} , 0 0 00 C7 : {1 , 1 , 1 } , 00 00 C8 : {1 , 1 , 1} , 00 00 0 (4.6) C9 : {1 , 1 , 1 } , 0 00 C10 : {1, 1 , 1 } . The three generations of matter fields are distinguished by their modular weights and transformation rules under A4 . If any two generations of fermion fields are assigned to the same singlet representation of A4 , their modular weight should be different. Once can also permute the above singlet assignments together with modular weights, for instance, the three generations of matter fields can be assigned to transform as 1, 10 and 100 or 100 , 1, 10 , this amounts to a redefinition of the fields. As a consequence, the predictions for the masses and mixing matrices of the quarks and leptons would keep invariant. Moreover, if neutrino masses are generated by the seesaw mechanism with two righthanded neutrinos which transform as singlets under A4 , then we have another two kinds of models: Type-VI : Type-VII : N1,2 ∼ 1(10 , 100 ) , 0 00 N1,2 ∼ 1(1 , 1 ) , F ∼ 3, F ∼ 3, (4.7) T ∼ 3, 0 00 T1,2,3 ∼ 1(1 , 1 ) . (4.8) As is generally true in two right-handed neutrino models, the lightest neutrino would be massless. The right-handed neutrinos N1 and N2 can transform in either the same or different manner under A4 , D1 : {1, 1} , D2 : {10 , 10 } , D3 : {100 , 100 } , D4 : {1, 10 } , D5 : {1, 100 } , D6 : {10 , 100 } . – 13 – (4.9) JHEP04(2021)239 Type-I : We see that either F or T are A4 singlets in Type-I, Type-II, Type-V and TypeVII models, and the mass hierarchies are achieved by introducing one free parameters for each generation. By comparison, the three generations of matter fields F and T are both assigned to transform as triplet 3 under the modular symmetry A4 in the Type-III, TypeIV and Type-VI models, thus the three generations of quark and lepton fields are treated on an equal footing. In order to reproduce the huge mass hierarchies among the quarks and leptons, fine-tuning of the coupling constants is necessary so that some comparable terms should cancel with each other to generate the small masses of the electron, up quark and down quark. Therefore the Type-III, Type-IV and Type-VI models are not favored by the current experiments, and more free parameters are needed to accommodate the mass hierarchies of quarks and leptons. For instance, we find a phenomenologically viable TypeIII model involving 40 real parameters, which is significantly larger than the number of parameters required for the Type-I, Type-II, Type-V and Type-VII models. In summary, we shall not analyse the Type-III, Type-IV and Type-VI models further in this paper since they are more fine tuned and require more parameters. 4.1 Type-I • Model I1 : (ρN , ρF , ρT1 , ρT2 , ρT3 ) = (3, 3, 100 , 1, 1) , (kN , kF , kT1 , kT2 , kT3 ) = (1, 1, 1, 1, 3) The modular invariant superpotentials for quark and lepton masses are of the following form, (2) Wν = αN1 Λ(N N )3S Y3 (2) (2) + αν1 (N F )3S Y3 H5 + αν2 (N F )3A Y3 H5 , (2) (2) (4) Wd = αd1 (F T1 )3 Y3 H5 + αd2 (F T2 )3 Y3 H5 + αd3 (F T3 )3 Y3 H5 (2) (2) (4) + αd0 1 (F T1 )3 Y3 H45 + αd0 2 (F T2 )3 Y3 H45 + αd0 3 (F T3 )3 Y3 H45 , (4) (4) (6) Wu = αu1 (T1 T3 )100 Y10 H5 + αu2 (T2 T3 )1 Y1 H5 + αu3 (T3 T3 )1 Y1 H5 . – 14 – (4.10) JHEP04(2021)239 Note that N1 and N2 are assigned to the same A4 singlet for the cases of D1,2,3 and they should carry different modular weights to be distinguished. For the Type-VII models, the three generations of the ten-plets T1 , T2 and T3 have ten possible representation assignments as shown in eq. (4.6). For each type of models, we comprehensively scan over the weights and representations of N , F and T . The requirement that each term of the superpotential W has vanishing total weight entails the modular weights of the matter fields N , F and T are integers. We neglect these cases that one column or one row of the fermion mass matrix is vanishing, since at least one fermion would be massless. A numerical analysis is performed for each model, and the strategy of numerical analysis is discussed in section 5. In the following, we present some benchmark models which can give acceptable masses and mixing parameters of both quark and leptons for certain values of the input parameters. The assignments of the matter fields for these viable models are summarized in table 2. Type-I #P (ρN , ρF , ρT1 , ρT2 , ρT3 ) kN kF I1 18 (3, 3, 100 , 1, 1) 1 1 (kT1 , kT2 , kT3 ) (1,1,3) I2 22 (3, 3, 100 , 10 , 10 ) 0 2 (2,0,4) I3 22 (3, 3, 100 , 100 , 10 ) 0 2 (0,2,4) I4 24 (3, 3, 100 , 1, 1) 0 2 (0,0,4) I5 24 (3, 3, 100 , 1, 1) 0 2 (0,2,4) I6 24 (3, 3, 100 , 1, 1) 0 2 (2,2,4) I7 24 (3, 3, 10 , 1, 100 ) 0 2 (0,0,4) I8 24 (3, 3, 100 , 1, 10 ) 2 0 (2,2,2) I9 24 (3, 3, 100 , 1, 10 ) 0 2 (2,2,4) I10 24 (3, 3, 10 , 10 , 100 ) 0 2 (0,2,4) I11 24 (3, 3, 100 , 10 , 10 ) 0 2 (2,2,4) I12 24 (3, 3, 100 , 100 , 10 ) 0 2 (2,4,2) I13 24 (3, 3, 100 , 1, 10 ) 2 0 (2,2,4) I14 24 (3, 3, 100 , 1, 10 ) 2 0 (2,4,4) 24 (3, 3, 100 , 10 , 10 ) 2 0 (2,2,4) 24 (3, 3, 100 , 100 , 10 ) 2 0 (2,4,2) I17 24 (3, 3, 100 , 100 , 10 ) 2 0 (2,4,4) Type-II #P (ρN , ρT , ρF1 , ρF2 , ρF3 ) kN (kF1 , kF2 , kF3 ) kT II 1 24 (3, 3, 1, 100 , 10 ) 1 (1,3,1) 3 II 2 24 (3, 3, 100 , 100 , 1) 1 (1,3,1) 3 Type-V #P (ρF , ρN1 , ρN2 , ρN3 , ρT1 , ρT2 , ρT3 ) (kN1 , kN2 , kN3 ) kF (kT1 , kT2 , kT3 ) V1 22 (3, 1, 1, 100 , 100 , 1, 1) (1,3,3) 1 (1,1,3) V2 22 (3, 1, 100 , 10 , 100 , 1, 1) (3,1,3) 1 (1,1,3) V3 24 (3, 1, 100 , 10 , 100 , 1, 10 ) (0,2,2) 2 (2,2,2) V4 24 (3, 1, 100 , 10 , 100 , 1, 10 ) (4,2,2) 0 (2,2,2) V5 24 (3, 1, 100 , 10 , 100 , 10 , 10 ) (4,2,2) 0 (2,2,4) V6 24 (3, 1, 100 , 10 , 100 , 100 , 10 ) (4,2,2) 0 (2,4,2) V7 24 (3, 1, 100 , 10 , 100 , 100 , 10 ) (4,2,2) 0 (2,4,4) V8 24 (3, 10 , 10 , 1, 100 , 1, 10 ) (2,4,2) 0 (2,2,2) V9 24 (3, 10 , 10 , 1, 100 , 10 , 10 ) (2,4,2) 0 (2,2,4) V10 24 (3, 10 , 10 , 1, 100 , 100 , 10 ) (2,4,2) 0 (2,4,2) V11 24 (3, 10 , 10 , 1, 100 , 100 , 10 ) (2,4,2) 0 (2,4,4) V12 24 (3, 100 , 100 , 1, 100 , 1, 10 ) (2,4,2) 0 (2,2,2) V13 24 (3, 100 , 100 , 1, 100 , 1, 10 ) (2,4,2) 0 (2,4,4) V14 24 (3, 100 , 100 , 1, 100 , 1, 1) (2,4,2) 0 (2,2,4) V15 24 (3, 1, 100 , 10 , 10 , 100 , 100 ) (0,0,0) 2 (0,0,4) V16 24 (3, 1, 100 , 10 , 10 , 100 , 1) (0,0,0) 2 (0,0,4) V17 24 (3, 1, 100 , 10 , 10 , 100 , 1) (0,0,0) 4 (0,0,4) V18 24 (3, 1, 100 , 10 , 10 , 100 , 1) (2,0,0) 2 (0,0,4) V19 24 (3, 1, 100 , 10 , 10 , 100 , 100 ) (0,0,0) 4 (0,0,4) V20 24 (3, 1, 100 , 10 , 10 , 100 , 100 ) (2,0,0) 2 (0,0,4) V21 24 (3, 1, 100 , 10 , 100 , 10 , 10 ) (0,0,0) 2 (2,0,4) V22 24 (3, 1, 100 , 10 , 100 , 10 , 10 ) (2,0,0) 2 (2,0,4) V23 24 (3, 1, 100 , 10 , 100 , 100 , 10 ) (0,0,0) 2 (0,2,4) V24 24 (3, 1, 100 , 10 , 100 , 100 , 10 ) (2,0,0) 2 (0,2,4) V25 24 (3, 10 , 10 , 100 , 10 , 100 , 1) (0,2,0) 2 (0,0,4) V26 24 (3, 10 , 10 , 100 , 10 , 100 , 100 ) (0,2,0) 2 (0,0,4) V27 24 (3, 10 , 10 , 100 , 100 , 10 , 10 ) (0,2,0) 2 (2,0,4) V28 24 (3, 10 , 10 , 100 , 100 , 100 , 10 ) (0,2,0) 2 (0,2,4) Type-VII #P (ρF , ρN1 , ρN2 , ρT1 , ρT2 , ρT3 ) (kN1 , kN2 ) kF (kT1 , kT2 , kT3 ) VII 1 22 (3, 1, 100 , 100 , 1, 1) (1,3) 3 (1,1,3) VII 2 22 (3, 10 , 10 , 100 , 1, 1) (1,3) 3 (1,1,3) VII 3 22 (3, 10 , 100 , 100 , 1, 1) (1,3) 3 (1,1,3) VII 4 23 (3, 1, 1, 100 , 100 , 10 ) (0,2) 2 (0,2,4) VII 5 23 (3, 1, 10 , 100 , 100 , 10 ) (0,2) 2 (0,2,4) VII 6 23 (3, 1, 10 , 100 , 100 , 10 ) (2,2) 2 (0,2,4) Table 2. Summary of phenomenologically viable SU(5) GUT models based on A4 modular symmetry with less free parameters. Here the real and imaginary part of τ are taken as free parameters. – 15 – JHEP04(2021)239 I15 I16 The right-handed neutrino mass matrix and the Yukawa matrix are given by  mνM (2) 2αN1 Y3,1  (2)  = Λ  −αN1 Y3,3 (2) −αN1 Y3,2  (2) (2) − αN1 Y3,2 (2)  − αN1 Y3,1  , (2) 2αN1 Y3,3 (2) 2αν1 Y3,1  (2) ν  Y5 =  − (αν1 + αν2 ) Y3,3 (2) − (αν1 − αν2 ) Y3,2 (4)  (4)  (2) αd2 Y3,1 (2) αd2 Y3,3 (2) αd2 Y3,2 (2) αd3 Y3,1 (4)  αd3 Y3,3  , (4) αd3 Y3,2 0  u Y5 =  0  (4) α u1 Y 1 0 0 0 (4) α u2 Y 1 α u1 Y 1 0 (4)  α u2 Y 1  . (6) α u3 Y 1 α Y  d1 3,2 (2) d  Y5 =  αd1 Y3,1 (2) αd1 Y3,3  (2) − (αν1 − αν2 ) Y3,3 (2) 2αν1 Y3,2 (2) − (αν1 + αν2 ) Y3,1  − (αν1 + αν2 ) Y3,2 (2)  − (αν1 − αν2 ) Y3,1  , (2) 2αν1 Y3,3 (4.11) Since both H5 and H45 are assumed to be invariant under A4 and their modular weights d are of the same form except that the couplings α are replaced are vanishing, Y5d and Y45 di d here and by αd0 i . Hence we shall not give the explicit form of the Yukawa coupling Y45 below. The parameters αN1 , αν1 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and αν2 , αd0 2 , αd0 3 , αu1 and αu2 are generically complex. • Model I2 : (ρN , ρF , ρT1 , ρT2 , ρT3 ) = (3, 3, 100 , 10 , 10 ) , (kN , kF , kT1 , kT2 , kT3 ) = (0, 2, 2, 0, 4) We can read out the superpotential for quark and leptons as follows, (2) (2) Wν = αN1 Λ(N N )1 +αν1 (N F )3S Y3 H5 +αν2 (N F )3A Y3 H5 , (4) (2) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (4) (2) (6) (4) (8) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (6) Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T3 )100 Y10 H5 +αu3 (T3 T3 )100 Y10 H5 . (4.12) The right-handed neutrino mass matrix and the Yukawa matrix are given by  mνM α N1  = Λ 0 0 0 0 α N1 0  α N1  , 0  αd1 Y3,2 (4) αd2 Y3,3 (4) Y5d =   αd1 Y3,1 (4) αd1 Y3,3 αd2 Y3,2    0  Y5u =  0  (6) α u1 Y 1 (2) αd3 Y3I,3 + αd4 Y3II,3 (2) (6) (6) αd3 Y3I,2 + αd4 Y3II,2  , (2) (6) (6)  (6) (6) αd2 Y3,1 αd3 Y3I,1 + αd4 Y3II,1 0 0 (4) α u2 Y 1 0 α u1 Y 1 (4)  α u2 Y 1 0  . (8) α u3 Y 1 0 – 16 –  (6)  (4.13) JHEP04(2021)239   (2) − αN1 Y3,3 (2) 2αN1 Y3,2 (2) − αN1 Y3,1 The Y5ν matrix is in common with that of the model I1 . The parameters αN1 , αν1 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and αν2 , αd4 , αd0 2 , αd0 3 , αd0 4 , αu1 and αu2 are generically complex. • Model I3 : (ρN , ρF , ρT1 , ρT2 , ρT3 ) = (3, 3, 100 , 100 , 10 ) , (kN , kF , kT1 , kT2 , kT3 ) = (0, 2, 0, 2, 4) The modular invariant superpotential is of the following form: (2) (2) Wν = αN1 Λ(N N )1 +αν1 (N F )3S Y3 H5 +αν2 (N F )3A Y3 H5 , (2) (4) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (4) (6) (6) (4) (6) (8) Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T3 )1 Y1 H5 +αu3 (T3 T3 )100 Y10 H5 , (4.14) which leads to αd1 Y3,2 (2) αd2 Y3,2 (2) Y5d =   αd1 Y3,1 (2) αd1 Y3,3 αd2 Y3,1    0  u  Y5 =  0 (4) α u1 Y 1 (4) αd3 Y3I,3 + αd4 Y3II,3 (4) (6) (6) αd3 Y3I,2 + αd4 Y3II,2  , (4) (6) (6)   (6) (6) αd2 Y3,3 αd3 Y3I,1 + αd4 Y3II,1 0 0 (6) α u2 Y 1 α u1 Y 1 (6)  α u2 Y 1  . (8) α u3 Y 1 0 (4)  (4.15) The right-handed neutrino mass matrix MN matrix is the same as that of the model I2 , and Y5ν is the same as that of the model I1 . The phases of the parameters αN1 , αν1 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be removed by field redefinition, and the remaining parameters αν2 , αd4 , αd0 2 , αd0 3 , αd0 4 , αu1 and αu2 are complex. • Model I4 : (ρN , ρF , ρT1 , ρT2 , ρT3 ) = (3, 3, 100 , 1, 1) , (kN , kF , kT1 , kT2 , kT3 ) = (0, 2, 0, 0, 4) The quark and lepton masses are described by the following superpotential: (2) (2) Wν = αN1 Λ(N N )1 +αν1 (N F )3S Y3 H5 +αν2 (N F )3A Y3 H5 , (2) (2) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (2) (2) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (4) (4) (8) Wu = αu1 (T1 T3 )100 Y10 H5 +αu2 (T2 T2 )1 H5 +αu3 (T2 T3 )1 Y1 H5 +αu4 (T3 T3 )1 Y1 H5 . (4.16) The right-handed neutrino mass matrix and the Yukawa matrix are given by αd1 Y3,2 (2) αd2 Y3,1 (2) Y5d =   αd1 Y3,1 (2) αd1 Y3,3 αd2 Y3,3    0  Y5u =  0  (4) α u1 Y 1 0 (2) αd3 Y3I,1 + αd4 Y3II,1 (2) (6) (6) αd3 Y3I,3 + αd4 Y3II,3  , (2) (6) (6)  (6) (6) αd2 Y3,2 αd3 Y3I,2 + αd4 Y3II,2 0 α u2 (4) α u3 Y 1 α u1 Y 1 0 (4)  α u3 Y 1  . (8) α u4 Y 1 – 17 –  (4)  (4.17) JHEP04(2021)239 (2) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , The matrices MN and Y5ν coincide with those of the model I3 as well. The phases of the parameters αN1 , αν1 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be removed by field redefinition, and the other parameters are complex. • Model I5 : (ρN , ρF , ρT1 , ρT2 , ρT3 ) = (3, 3, 100 , 1, 1) , (kN , kF , kT1 , kT2 , kT3 ) = (0, 2, 0, 2, 4) The modular invariant superpotentials for quark and lepton masses are of the following form, (2) (2) (4) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (2) (4) (4) (4) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (6) (8) Wu = αu1 (T1 T3 )100 Y10 H5 +αu2 (T2 T2 )1 Y1 H5 +αu3 (T2 T3 )1 Y1 H5 +αu4 (T3 T3 )1 Y1 H5 . (4.18) The right-handed neutrino mass matrix and the Yukawa coupling matrices are predicted to be αd1 Y3,2 (2) αd2 Y3,1 (2) Y5d =   αd1 Y3,1 (2) αd1 Y3,3 αd2 Y3,3    0  Y5u =  0  (4) α u1 Y 1 0 (4) αd3 Y3I,1 + αd4 Y3II,1 (4) (6) (6) αd3 Y3I,3 + αd4 Y3II,3  , (4) (6) (6)   (6) (6) αd2 Y3,2 αd3 Y3I,2 + αd4 Y3II,2 0 (4) α u2 Y 1 (6) α u3 Y 1 α u1 Y 1 0 (6)  α u3 Y 1  . (8) α u4 Y 1 (4)  (4.19) The matrices MN and Y5ν are the same as the corresponding ones of the model I3 as well. The parameters αN1 , αν1 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and the other parameters are complex. • Model I6 : (ρN , ρF , ρT1 , ρT2 , ρT3 ) = (3, 3, 100 , 1, 1) , (kN , kF , kT1 , kT2 , kT3 ) = (0, 2, 2, 2, 4) The quark and lepton masses are described by the following superpotential: (2) (2) Wν = αN1 Λ(N N )1 +αν1 (N F )3S Y3 H5 +αν2 (N F )3A Y3 H5 , (4) (4) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (4) (4) (4) (4) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (6) (8) Wu = αu1 (T1 T2 )100 Y10 H5 +αu2 (T2 T2 )1 Y1 H5 +αu3 (T2 T3 )1 Y1 H5 +αu4 (T3 T3 )1 Y1 H5 , (4.20) – 18 – JHEP04(2021)239 (2) Wν = αN1 Λ(N N )1 +αν1 (N F )3S Y3 H5 +αν2 (N F )3A Y3 H5 , which gives rise to αd1 Y3,2 (4) αd2 Y3,1 (4) Y5d =   αd1 Y3,1 (4) αd1 Y3,3 αd2 Y3,3    0  u  Y5 =  αu1 Y1(4) 0 0 (4) αd3 Y3I,1 + αd4 Y3II,1 (4) (6) (6) αd3 Y3I,3 + αd4 Y3II,3  , (4) αd2 Y3,2 (4) α u1 Y 1 0 (4) α u2 Y 1 (6) α u3 Y 1 (6) (6)   (6) (6) αd3 Y3I,2 + αd4 Y3II,2  0 (6)  α u3 Y 1  . (8) α u4 Y 1 (4.21) • Model I7 : (ρN , ρF , ρT1 , ρT2 , ρT3 ) = (3, 3, 10 , 1, 100 ) , (kN , kF , kT1 , kT2 , kT3 ) = (0, 2, 0, 0, 4) The modular invariant superpotential is of the following form: (2) (2) Wν = αN1 Λ(N N )1 +αν1 (N F )3S Y3 H5 +αν2 (N F )3A Y3 H5 , (2) (2) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (2) (2) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (4) (4) (8) Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T2 )1 H5 +αu3 (T2 T3 )100 Y10 H5 +αu4 (T3 T3 )10 Y100 H5 . (4.22) The right-handed neutrino mass matrix and the Yukawa matrix are given by αd1 Y3,3 (2) αd2 Y3,1 (2) Y5d =   αd1 Y3,2 (2) αd1 Y3,1 αd2 Y3,3    0  u Y5 =  0  (4) α u1 Y 1 (2) αd3 Y3I,2 + αd4 Y3II,2 (2) (6) (6) αd3 Y3I,1 + αd4 Y3II,1  , (2) (6) (6)   (6) (6) αd2 Y3,2 αd3 Y3I,3 + αd4 Y3II,3 0 α u2 (4) α u3 Y 1 0 α u1 Y 1 (4)  α u3 Y 1 0  . (8) αu4 Y100 (4)  (4.23) The matrices MN and Y5ν coincide with those of the model I3 as well. The parameters αN1 , αν1 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and the other parameters are complex. • Model I8 : (ρN , ρF , ρT1 , ρT2 , ρT3 ) = (3, 3, 100 , 1, 10 ) , (kN , kF , kT1 , kT2 , kT3 ) = (2, 0, 2, 2, 2) The modular invariant superpotential is of the following form: (4) (4) (4) (2) Wν = αN1 Λ(N N )1 Y1 +αN2 Λ(N N )100 Y10 +αN3 Λ(N N )3S Y3 +αν1 (N F )3S Y3 H5 (2) +αν2 (N F )3A Y3 H5 , (2) (2) (2) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (2) (4) (4) (2) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (4) (4) Wu = αu1 (T1 T2 )100 Y10 H5 +αu2 (T1 T3 )1 Y1 H5 +αu3 (T2 T2 )1 Y1 H5 +αu4 (T3 T3 )100 Y10 H5 . (4.24) – 19 – JHEP04(2021)239 The matrices MN and Y5ν are in common with those of the model I3 as well. The parameters αN1 , αν1 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and αν2 , αd4 , αd0 2 , αd0 3 , αd0 4 , αu1 , αu2 and αu4 are complex. The right-handed neutrino mass matrix and the Yukawa matrix are given by  mνM (4) (4) α Y + 2αN3 Y3,1  N1 1 (4)  = Λ −αN3 Y3,3 (4) (4) αN2 Y10 − αN3 Y3,2  (4)  (4)  αd2 Y3,1 (2) αd2 Y3,3 (2) αd2 Y3,2 (2) αd3 Y3,3 (2)  αd3 Y3,2  , (2) αd3 Y3,1 0  (4) Y5u =   α u1 Y 1 0 (4) α u2 Y 1 α u1 Y 1 0 (4) α u3 Y 1 0 (4) α u2 Y 1  . 0  (4) α u4 Y 1 0  (4)  αN2 Y10 − αN3 Y3,2 (4) (4)  αN1 Y1 − αN3 Y3,1  , (4) 2αN3 Y3,3 (4.25) The Y5ν matrix coincide with those of the model I2 as well. The phases of the parameters αN1 , αν1 , αd1 , αd2 , αd3 , αd0 1 and αu3 are unphysical, and the remaining parameters are complex. • Model I9 : (ρN , ρF , ρT1 , ρT2 , ρT3 ) = (3, 3, 100 , 1, 10 ) , (kN , kF , kT1 , kT2 , kT3 ) = (0, 2, 2, 2, 4) We can read out the superpotentials relevant to quark and lepton masses (2) (2) Wν = αN1 Λ(N N )1 +αν1 (N F )3S Y3 H5 +αν2 (N F )3A Y3 H5 , (4) (4) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (4) (4) (4) (6) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (4) (8) Wu = αu1 (T1 T2 )100 Y10 H5 +αu2 (T1 T3 )1 Y1 H5 +αu3 (T2 T2 )1 Y1 H5 +αu4 (T3 T3 )100 Y10 H5 , (4.26) which lead to αd1 Y3,2 (4) αd2 Y3,1 (4) Y5d =   αd1 Y3,1 (4) αd1 Y3,3 αd2 Y3,3    0  u  Y5 =  αu1 Y1(4) 0 (6) α u2 Y 1 (4) αd3 Y3I,3 + αd4 Y3II,3 (4) (6) (6) αd3 Y3I,2 + αd4 Y3II,2  , (4) αd2 Y3,2 (4) α u1 Y 1 0 (4) α u3 Y 1 0 (6) (6)   (6) (6) αd3 Y3I,1 + αd4 Y3II,1 (6)  α u2 Y 1  . 0  (8) α u4 Y 1 0 (4.27) The matrices MN and Y5ν are identical with those of the model I3 as well. The phases of the parameters αN1 , αν1 , αd1 , αd2 , αd3 , αd0 1 and αu3 are unphysical, and the other parameters are complex. – 20 – JHEP04(2021)239 (2) (2) α Y  d1 3,2 (2) d Y5 =   αd1 Y3,1 (2) αd1 Y3,3 (4) − αN3 Y3,3 (4) (4) αN2 Y10 + 2αN3 Y3,2 (4) (4) αN1 Y1 − αN3 Y3,1 • Model I10 : (ρN , ρF , ρT1 , ρT2 , ρT3 ) = (3, 3, 10 , 10 , 100 ) , (kN , kF , kT1 , kT2 , kT3 ) = (0, 2, 0, 2, 4) We can read out the superpotential for quark and leptons as follows, (2) (2) Wν = αN1 Λ(N N )1 + αν1 (N F )3S Y3 H5 + αν2 (N F )3A Y3 H5 , (2) (4) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 + αd2 (F T2 )3 Y3 H5 + αd3 (F T3 )3 Y3,I H5 + αd4 (F T3 )3 Y3,II H5 (2) (4) (6) (6) + αd0 1 (F T1 )3 Y3 H45 + αd0 2 (F T2 )3 Y3 H45 + αd0 3 (F T3 )3 Y3,I H45 + αd0 4 (F T3 )3 Y3,II H45 , (4) (4) (6) (8) Wu = αu1 (T1 T3 )1 Y1 H5 + αu2 (T2 T2 )100 Y10 H5 + αu3 (T2 T3 )1 Y1 H5 + αu4 (T3 T3 )10 Y100 H5 . (4.28) αd1 Y3,3 (2) αd2 Y3,3 (2) Y5d =   αd1 Y3,2 (2) αd1 Y3,1 αd2 Y3,2     Y5u =   0 0 (4) α u1 Y 1 (4) αd3 Y3I,2 + αd4 Y3II,2 (4) (6) (6) αd3 Y3I,1 + αd4 Y3II,1  , (4) αd2 Y3,1 0 (4) α u2 Y 1 0 (6) α u3 Y 1 (6) (6)   (6) (6) αd3 Y3I,3 + αd4 Y3II,3 (4)  α u1 Y 1 (6)  α u3 Y 1  . (8) αu4 Y100 (4.29) The matrices MN and Y5ν are the same as the corresponding ones of the model I3 as well. The parameters αN1 , αν1 , αd1 , αd2 , αd3 , αd0 1 and αu3 are real, and the remaining parameters are complex. • Model I11 : (ρN , ρF , ρT1 , ρT2 , ρT3 ) = (3, 3, 100 , 10 , 10 ) , (kN , kF , kT1 , kT2 , kT3 ) = (0, 2, 2, 2, 4) The modular invariant superpotential for quark and lepton mass is given by (2) (2) Wν = αN1 Λ(N N )1 +αν1 (N F )3S Y3 H5 +αν2 (N F )3A Y3 H5 , (4) (4) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (4) (4) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (4) (6) (4) (8) Wu = αu1 (T1 T2 )1 Y1 H5 +αu2 (T1 T3 )1 Y1 H5 +αu3 (T2 T2 )100 Y10 H5 +αu4 (T3 T3 )100 Y10 H5 . (4.30) The right-handed neutrino mass matrix and the Yukawa matrix are given by αd1 Y3,2 (4) αd2 Y3,3 (4) Y5d =   αd1 Y3,1 αd2 Y3,2 αd1 Y3,3 αd2 Y3,1 0  u  Y5 =  αu1 Y1(4) (6) α u2 Y 1 α u1 Y 1 (4) α u3 Y 1 0 0   (4)  (4) αd3 Y3I,3 + αd4 Y3II,3 (4) (6) (6) αd3 Y3I,2 + αd4 Y3II,2  , (4) (4) (6) (6)   (6) (6) αd3 Y3I,1 + αd4 Y3II,1 (6)  α u2 Y 1  . 0  (8) α u4 Y 1 0 (4.31) The matrices MN and Y5ν are identical with those of the model I3 as well. The parameters αN1 , αν1 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and the remaining parameters are complex. – 21 – JHEP04(2021)239 The right-handed neutrino mass matrix and the Yukawa coupling matrices are predicted to be • Model I12 : (ρN , ρF , ρT1 , ρT2 , ρT3 ) = (3, 3, 100 , 100 , 10 ) , (kN , kF , kT1 , kT2 , kT3 ) = (0, 2, 2, 4, 2) The quark and lepton masses are described by the following superpotential: (2) (2) Wν = αN1 Λ(N N )1 +αν1 (N F )3S Y3 H5 +αν2 (N F )3A Y3 H5 , (4) (6) (6) (4) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3,I H5 +αd4 (F T2 )3 Y3,II H5 +αd3 (F T3 )3 Y3 H5 (4) (6) (6) (4) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3,I H45 +αd0 4 (F T2 )3 Y3,II H45 +αd0 3 (F T3 )3 Y3 H45 , (4) (8) (6) (4) Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T2 )10 Y100 H5 +αu3 (T2 T3 )1 Y1 H5 +αu4 (T3 T3 )100 Y10 H5 , (4.32) αd1 Y3,2 (4) αd2 Y3I,2 + αd4 Y3II,2 (4) Y5d =   αd1 Y3,1 (4) αd1 Y3,3 αd2 Y3I,1 + αd4 Y3II,1    0  Y5u =  0  (4) α u1 Y 1 (6) (6) αd3 Y3,3 (4) (6) (6) (6) (6) (4) αd3 Y3,2  ,  (4) αd2 Y3I,3 + αd4 Y3II,3 0 (8) αu2 Y100 (6) α u3 Y 1 (4)  αd3 Y3,1  α u1 Y 1 (6)  α u3 Y 1  . (4) α u4 Y 1 0 (4.33) The matrices MN and Y5ν are identical with the corresponding ones of the model I3 as well. The phases of the parameters αN1 , αν1 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be removed by field redefinition, and the remaining parameters are complex. • Model I13 : (ρN , ρF , ρT1 , ρT2 , ρT3 ) = (3, 3, 100 , 1, 10 ) , (kN , kF , kT1 , kT2 , kT3 ) = (2, 0, 2, 2, 4) The modular invariant superpotential is of the following form: (4) (4) (4) (2) Wν = αN1 Λ(N N )1 Y1 +αN2 Λ(N N )100 Y10 +αN3 Λ(N N )3S Y3 +αν1 (N F )3S Y3 H5 (2) +αν2 (N F )3A Y3 H5 , (2) (2) (4) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (2) (4) (6) (4) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (4) (8) Wu = αu1 (T1 T2 )100 Y10 H5 +αu2 (T1 T3 )1 Y1 H5 +αu3 (T2 T2 )1 Y1 H5 +αu4 (T3 T3 )100 Y10 H5 . (4.34) which gives rise to  (2) α Y  d1 3,2 (2) d  Y5 =  αd1 Y3,1 (2) αd1 Y3,3 (2) αd2 Y3,1 (2) αd2 Y3,3 (2) αd2 Y3,2 (4)  αd3 Y3,3 (4)  αd3 Y3,2  . (4) αd3 Y3,1 (4.35) The MN matrix coincides with that of the model I8 . The Y5ν matrix coincides with that of the model I1 . The Y5u matrix is identical with the corresponding one of the model I9 . The parameters αN3 , αν1 , αd1 , αd2 , αd3 , αd0 1 and αu3 are real, and the remaining parameters αN1 , αN2 , αν2 , αd0 2 , αd0 3 , αu1 , αu2 and αu4 are complex. – 22 – JHEP04(2021)239 which gives rise to • Model I14 : (ρN , ρF , ρT1 , ρT2 , ρT3 ) = (3, 3, 100 , 1, 10 ) , (kN , kF , kT1 , kT2 , kT3 ) = (2, 0, 2, 4, 4) We can read out the superpotential for the fermion masses as, (4) (4) (4) (2) Wν = αN1 Λ(N N )1 Y1 +αN2 Λ(N N )100 Y10 +αN3 Λ(N N )3S Y3 +αν1 (N F )3S Y3 H5 (2) +αν2 (N F )3A Y3 H5 , (2) (4) (4) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (4) (4) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (6) (8) (8) (8) which gives rise to  (4)  (6)  (2) αd2 Y3,1 (4) αd2 Y3,3 (4) αd2 Y3,2 (4) αd3 Y3,3 (4)  αd3 Y3,2  , (4) αd3 Y3,1 0  u  Y5 =  0 (6) α u1 Y 1 0 (8) α u2 Y 1 (8) αu3 Y100 α u1 Y 1 (8)  αu3 Y100  . (8) α u4 Y 1 0 α Y  d1 3,2 (2) d  Y5 =  αd1 Y3,1 (2) αd1 Y3,3  (4.37) The matrices MN and Y5ν are identical with those of the models I8 and I1 respectively. The parameters αN3 , αν1 , αd1 , αd2 , αd3 , αd0 1 and αu3 can taken to be real without loss of generality, while αN1 , αN2 , αν2 , αd0 2 , αd0 3 , αu1 , αu2 and αu4 are generically complex. • Model I15 : (ρN , ρF , ρT1 , ρT2 , ρT3 ) = (3, 3, 100 , 10 , 10 ) , (kN , kF , kT1 , kT2 , kT3 ) = (2, 0, 2, 2, 4) The modular invariant superpotential for quark and lepton mass is given by (4) (4) (4) (2) Wν = αN1 Λ(N N )1 Y1 +αN2 Λ(N N )100 Y10 +αN3 Λ(N N )3S Y3 +αν1 (N F )3S Y3 H5 (2) +αν2 (N F )3A Y3 H5 , (2) (2) (4) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (2) (4) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (4) (6) (4) (8) Wu = αu1 (T1 T2 )1 Y1 H5 +αu2 (T1 T3 )1 Y1 H5 +αu3 (T2 T2 )100 Y10 H5 +αu4 (T3 T3 )100 Y10 H5 , (4.38) which leads to  (2) α Y  d1 3,2 (2) Y5d =   αd1 Y3,1 (2) αd1 Y3,3 (2) αd2 Y3,3 (2) αd2 Y3,2 (2) αd2 Y3,1 (4)  αd3 Y3,3 (4)  αd3 Y3,2  . (4) αd3 Y3,1 (4.39) The MN matrix coincides with the corresponding one of the model I8 . The Y5ν matrix coincides with that of the model I1 . The Y5u matrix is identical with that of the model I11 . The parameters αN3 , αν1 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and the remaining parameters are complex. – 23 – JHEP04(2021)239 Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T2 )1 Y1 H5 +αu3 (T2 T3 )10 Y100 H5 +αu4 (T3 T3 )100 Y10 H5 . (4.36) • Model I16 : (ρN , ρF , ρT1 , ρT2 , ρT3 ) = (3, 3, 100 , 100 , 10 ) , (kN , kF , kT1 , kT2 , kT3 ) = (2, 0, 2, 4, 2) We can read out the superpotentials relevant to quark and lepton masses (4) (4) (4) (2) Wν = αN1 Λ(N N )1 Y1 +αN2 Λ(N N )100 Y10 +αN3 Λ(N N )3S Y3 +αν1 (N F )3S Y3 H5 (2) +αν2 (N F )3A Y3 H5 , (2) (4) (2) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (4) (2) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (4) (8) (6) (4) The right-handed neutrino mass matrix and the Yukawa matrix are given by  (2) (4) α Y  d1 3,2 (2) d  Y5 =  αd1 Y3,1 (2) αd1 Y3,3 αd2 Y3,2 (4) αd2 Y3,1 (4) αd2 Y3,3  (2) αd3 Y3,3 (2)  αd3 Y3,2  . (2) αd3 Y3,1 (4.41) The MN matrix is the same as the corresponding one of the model I8 . The Y5ν matrix is in common with the corresponding one of the model I1 . The Y5u matrix coincides with the corresponding one of the model I12 . The parameters αN3 , αν1 , αd1 , αd2 , αd3 , αd0 1 and αu3 can taken to be real without loss of generality, while αN1 , αN2 , αν2 , αd0 2 , αd0 3 , αu1 , αu2 and αu4 are generically complex. • Model I17 : (ρN , ρF , ρT1 , ρT2 , ρT3 ) = (3, 3, 100 , 100 , 10 ) , (kN , kF , kT1 , kT2 , kT3 ) = (2, 0, 2, 4, 4) We can read out the superpotential for the fermion masses as, (4) (4) (4) (2) Wν = αN1 Λ(N N )1 Y1 +αN2 Λ(N N )100 Y10 +αN3 Λ(N N )3S Y3 +αν1 (N F )3S Y3 H5 (2) +αν2 (N F )3A Y3 H5 , (2) (4) (4) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (4) (4) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (6) (8) (8) (8) Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T2 )10 Y100 H5 +αu3 (T2 T3 )1 Y1 H5 +αu4 (T3 T3 )100 Y10 H5 . (4.42) The right-handed neutrino mass matrix and the Yukawa matrix are given by  (4)  (6)  (2) αd2 Y3,2 (4) αd2 Y3,1 (4) αd2 Y3,3 (4) αd3 Y3,3 (4)  αd3 Y3,2  , (4) αd3 Y3,1 0  u  Y5 =  0 (6) α u1 Y 1 0 (8) αu2 Y100 (8) α u3 Y 1 α u1 Y 1 (8)  α u3 Y 1  . (8) α u4 Y 1 0 α Y  d1 3,2 (2) d  Y5 =  αd1 Y3,1 (2) αd1 Y3,3  (4.43) The matrices MN and Y5ν coincide with the corresponding ones of the model I14 as well. The parameters αN3 , αν1 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and the other parameters are complex. – 24 – JHEP04(2021)239 Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T2 )10 Y100 H5 +αu3 (T2 T3 )1 Y1 H5 +αu4 (T3 T3 )100 Y10 H5 . (4.40) 4.2 Type-II • Model II 1 : (ρN , ρF1 , ρF2 , ρF3 , ρT ) = (3, 1, 100 , 10 , 3) , (kN , kF1 , kF2 , kF3 , kT ) = (1, 1, 3, 1, 3) The quark and lepton masses are described by the following superpotential: (2) (2) (4) (2) Wν = αN1 Λ(N N )3S Y3 +αν1 (N F1 )3 Y3 H5 +αν2 (N F2 )3 Y3 H5 +αν3 (N F3 )3 Y3 H5 , (4) (6) (6) (4) Wd = αd1 (F1 T )3 Y3 H5 +αd2 (F2 T )3 Y3,I H5 +αd4 (F2 T )3 Y3,II H5 +αd3 (F3 T )3 Y3 H5 (4) (6) (6) (4) +αd0 1 (F1 T )3 Y3 H45 +αd0 2 (F2 T )3 Y3,I H45 +αd0 4 (F2 T )3 Y3,II H45 +αd0 3 (F3 T )3 Y3 H45 , (6) (6) (6) (4.44) The right-handed neutrino mass matrix and the Yukawa matrix are given by  (2) αν Y  1 3,1 (2) Y5ν =   αν1 Y3,3 (2) αν1 Y3,2  (4) (2) αν2 Y3,2 (4) αν2 Y3,1 (4) αν2 Y3,3 (4) αd1 Y3,3 (6) (6) αd2 Y3I,1 +αd4 Y3II,1 (4) αd3 Y3,2 αd1 Y3,1  (6) (6) d  Y5 =  αd2 Y3I,2 +αd4 Y3II,2 (4) (4) αd3 Y3,3   αν3 Y3,3 (2)  αν3 Y3,2  , (2) αν3 Y3,1 (6) (6) (6) αu1 Y1 +2αu2 Y3I,1 +2αu3 Y3II,1 (6) Y5u =    (6)  (4) αd3 Y3,1 (6) (6) (6) (6) (6) −α Y −αu3 Y3II,3  u2 3I,3 (6) (6) −αu2 Y3I,2 −αu3 Y3II,2 (6) (6)  −αu2 Y3I,2 −αu3 Y3II,2 (6) 2 αu2 Y3I,2 +αu3 Y3II,2 −αu2 Y3I,3 −αu3 Y3II,3 (6) (4) αd1 Y3,2 (6) (6)  αd2 Y3I,3 +αd4 Y3II,3  , (6) αu1 Y1 −αu2 Y3I,1 −αu3 Y3II,1 (6) (6)  αu1 Y1 −αu2 Y3I,1 −αu3 Y3II,1  .  (6) (6) 2 αu2 Y3I,3 +αu3 Y3II,3  (4.45) The MN matrix coincides with the corresponding one of the model I1 . The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and the remaining parameters are complex. • Model II 2 : (ρN , ρF1 , ρF2 , ρF3 , ρT ) = (3, 100 , 100 , 1, 3) , (kN , kF1 , kF2 , kF3 , kT ) = (1, 1, 3, 1, 3) The modular invariant superpotential for quark and lepton mass is given by (2) (2) (4) (2) Wν = αN1 Λ(N N )3S Y3 +αν1 (N F1 )3 Y3 H5 +αν2 (N F2 )3 Y3 H5 +αν3 (N F3 )3 Y3 H5 , (4) (6) (6) (4) Wd = αd1 (F1 T )3 Y3 H5 +αd2 (F2 T )3 Y3,I H5 +αd4 (F2 T )3 Y3,II H5 +αd3 (F3 T )3 Y3 H5 (4) (6) (6) (4) +αd0 1 (F1 T )3 Y3 H45 +αd0 2 (F2 T )3 Y3,I H45 +αd0 4 (F2 T )3 Y3,II H45 +αd0 3 (F3 T )3 Y3 H45 , (6) (6) (6) Wu = αu1 (T T )1 Y1 H5 +αu2 (T T )3S Y3,I H5 +αu3 (T T )3S Y3,II H5 . (4.46) The right-handed neutrino mass matrix and the Yukawa matrix are given by  (2) αν Y  1 3,2 (2) ν  Y5 =  αν1 Y3,1 (2) αν1 Y3,3  (4) αν2 Y3,2 (4) αν2 Y3,1 (4) αν2 Y3,3 (4) αd1 Y3,2  (6) (6) d Y5 =   αd2 Y3I,2 + αd4 Y3II,2 (4) αd3 Y3,1 (2)  αν3 Y3,1 (2)  αν3 Y3,3  , (2) αν3 Y3,2 (4) αd1 Y3,1 (6) (6) αd2 Y3I,1 + αd4 Y3II,1 (4) αd3 Y3,3 – 25 – (4)  αd1 Y3,3 (6) (6)  αd2 Y3I,3 + αd4 Y3II,3  . (4) αd3 Y3,2 (4.47) JHEP04(2021)239 Wu = αu1 (T T )1 Y1 H5 +αu2 (T T )3S Y3,I H5 +αu3 (T T )3S Y3,II H5 . The matrices MN and Y5u are identical with those of the models I1 and II 1 respectively. The phases of the parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 are unphysical, and αd4 , αd0 2 , αd0 4 , αd0 3 , αu1 and αu2 are generically complex. 4.3 Type-V • Model V1 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 1, 100 , 3, 100 , 1, 1) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (1, 3, 3, 1, 1, 1, 3) We can read out the superpotential for quark and leptons as follows, (4) (6) (2) (4) (4) +αν2 (N2 F )3 Y3 H5 +αν3 (N3 F )3 Y3 H5 , (2) (2) (4) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (2) (4) (4) (4) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (6) Wu = αu1 (T1 T3 )100 Y10 H5 +αu2 (T2 T3 )1 Y1 H5 +αu3 (T3 T3 )1 Y1 H5 . (4.48) We can straightforwardly read out the right-handed neutrino mass matrix and the Yukawa matrices as follows,  mνM (4) 0  (4) = Λ  α N1 Y 1 (4) α N2 Y 1 0  (2) α N1 Y 1 (6) Y 1 α N3 0 (2) αν Y  1 3,1 (4) ν  Y5 =  αν2 Y3,1 (4) αν3 Y3,2 αν1 Y3,3 (4) αν2 Y3,3 (4) αν3 Y3,1 (4)  α N2 Y 1 0  , 0  0 (2)  αν1 Y3,2 (4)  αν2 Y3,2  . (4) αν3 Y3,3 (4.49) The matrices Y5d and Y5u are the same as those of the model I1 as well. The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and αN2 , αN3 , αd0 2 , αd0 3 , αu1 and αu2 are complex. • Model V2 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 100 , 10 , 3, 100 , 1, 1) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (3, 1, 3, 1, 1, 1, 3) The modular invariant superpotentials for quark and lepton masses are of the following form, (6) (4) (4) (4) Wν = αN1 Λ(N1 N1 )1 Y1 +αN2 Λ(N1 N2 )100 Y10 +αN3 Λ(N2 N3 )1 Y1 +αν1 (N1 F )3 Y3 H5 (2) (4) +αν2 (N2 F )3 Y3 H5 +αν3 (N3 F )3 Y3 H5 , (2) (2) (4) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (2) (4) (4) (4) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (6) Wu = αu1 (T1 T3 )100 Y10 H5 +αu2 (T2 T3 )1 Y1 H5 +αu3 (T3 T3 )1 Y1 H5 . – 26 – (4.50) JHEP04(2021)239 (4) Wν = αN1 Λ(N1 N2 )1 Y1 +αN2 Λ(N1 N3 )100 Y10 +αN3 Λ(N2 N2 )1 Y1 +αν1 (N1 F )3 Y3 H5 We can straightforwardly read out the right-handed neutrino mass matrix and the Yukawa matrices as follows,  mνM (6) Y α N1  1  = Λ  αN2 Y1(4) 0 0  (4) (4) α N2 Y 1 0 0 (4) α N3 Y 1 (4) αν1 Y3,3 (2) αν2 Y3,1 (4) αν3 Y3,2 (4)  αν1 Y3,2 (2)  αν2 Y3,3  . (4) αν3 Y3,1 (4.51) The matrices Y5d and Y5u coincide with the corresponding ones of the model V1 as well. The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and αN2 , αN3 , αd0 2 , αd0 3 , αu1 and αu2 are generically complex. • Model V3 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 100 , 10 , 3, 100 , 1, 10 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (0, 2, 2, 2, 2, 2, 2) The modular invariant superpotential for quark and lepton mass is given by (4) (4) (2) Wν = αN1 Λ(N1 N1 )1 +αN2 Λ(N2 N3 )1 Y1 +αN3 Λ(N3 N3 )100 Y10 +αν1 (N1 F )3 Y3 H5 (4) (4) +αν2 (N2 F )3 Y3 H5 +αν3 (N3 F )3 Y3 H5 , (4) (4) (4) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (4) (4) (4) (4) (4) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (4) (4) Wu = αu1 (T1 T2 )100 Y10 H5 +αu2 (T1 T3 )1 Y1 H5 +αu3 (T2 T2 )1 Y1 H5 +αu4 (T3 T3 )100 Y10 H5 , (4.52) which lead to  mνM 0 0 (4) α N2 Y 1 α N1   = Λ 0 0  0 (4)  α N2 Y 1  , (4) α N3 Y 1 0  (2) αν1 Y3,3 (4) αν2 Y3,1 (4) αν3 Y3,2 (2) αν1 Y3,2 (4)  αν2 Y3,3  , (4) αν3 Y3,1  (4) αd2 Y3,1 (4) αd2 Y3,3 (4) αd2 Y3,2 (4) αd3 Y3,3 (4)  αd3 Y3,2  . (4) αd3 Y3,1 αν Y  1 3,1 (4) ν  Y5 =  αν2 Y3,2 (4) αν3 Y3,3 α Y  d1 3,2 (4) d Y5 =   αd1 Y3,1 (4) αd1 Y3,3 (2)  (4)  (4.53) The Y5u matrix coincides with the corresponding one of the model I8 . The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and αN2 , αN3 , αd0 2 , αd0 3 , αu1 , αu2 and αu4 are complex. – 27 – JHEP04(2021)239 αν Y  1 3,1 (2) ν Y5 =   αν2 Y3,2 (4) αν3 Y3,3  0 (4)  α N3 Y 1  , 0 • Model V4 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 100 , 10 , 3, 100 , 1, 10 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (4, 2, 2, 0, 2, 2, 2) We can read out the superpotential for quark and leptons as follows, (8) (4) (4) (4) Wν = αN1 Λ(N1 N1 )1 Y1 +αN2 Λ(N2 N3 )1 Y1 +αN3 Λ(N3 N3 )100 Y10 +αν1 (N1 F )3 Y3 H5 (2) (2) +αν2 (N2 F )3 Y3 H5 +αν3 (N3 F )3 Y3 H5 , (2) (2) (2) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (2) (4) (4) (2) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (4) which gives rise to  mνM (8) 0 0 (4) α N2 Y 1 α Y  N1 1  = Λ 0 0  (4) (4) αν Y  1 3,1 (2) ν Y5 =   αν2 Y3,2 (2) αν3 Y3,3 αν1 Y3,3 (2) αν2 Y3,1 (2) αν3 Y3,2  0 (4)  α N2 Y 1  , (4) α N3 Y 1 0 (4)  αν1 Y3,2 (2)  αν2 Y3,3  . (2) αν3 Y3,1 (4.55) The matrices Y5d and Y5u are the same as those of the model I8 as well. The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can taken to be real without loss of generality, while αN2 , αN3 , αd0 2 , αd0 3 , αu1 , αu2 and αu4 are generically complex. • Model V5 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 100 , 10 , 3, 100 , 10 , 10 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (4, 2, 2, 0, 2, 2, 4) The quark and lepton masses are described by the following superpotential: (8) (4) (4) (4) Wν = αN1 Λ(N1 N1 )1 Y1 +αN2 Λ(N2 N3 )1 Y1 +αN3 Λ(N3 N3 )100 Y10 +αν1 (N1 F )3 Y3 H5 (2) (2) +αν2 (N2 F )3 Y3 H5 +αν3 (N3 F )3 Y3 H5 , (2) (2) (4) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (2) (4) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (4) (6) (4) (8) Wu = αu1 (T1 T2 )1 Y1 H5 +αu2 (T1 T3 )1 Y1 H5 +αu3 (T2 T2 )100 Y10 H5 +αu4 (T3 T3 )100 Y10 H5 . (4.56) The matrices MN and Y5ν are the same as those of the model V4 as well. The Y5d matrix is identical with the corresponding one of the model I15 . The Y5u matrix is in common with that of the model I11 . The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and the other parameters are complex. • Model V6 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 100 , 10 , 3, 100 , 100 , 10 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (4, 2, 2, 0, 2, 4, 2) – 28 – JHEP04(2021)239 (4) Wu = αu1 (T1 T2 )100 Y10 H5 +αu2 (T1 T3 )1 Y1 H5 +αu3 (T2 T2 )1 Y1 H5 +αu4 (T3 T3 )100 Y10 H5 . (4.54) The modular invariant superpotential for quark and lepton mass is given by (8) (4) (4) (4) Wν = αN1 Λ(N1 N1 )1 Y1 +αN2 Λ(N2 N3 )1 Y1 +αN3 Λ(N3 N3 )100 Y10 +αν1 (N1 F )3 Y3 H5 (2) (2) +αν2 (N2 F )3 Y3 H5 +αν3 (N3 F )3 Y3 H5 , (2) (4) (2) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (4) (2) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (4) (8) (6) (4) Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T2 )10 Y100 H5 +αu3 (T2 T3 )1 Y1 H5 +αu4 (T3 T3 )100 Y10 H5 . (4.57) • Model V7 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 100 , 10 , 3, 100 , 100 , 10 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (4, 2, 2, 0, 2, 4, 4) The modular invariant superpotentials for quark and lepton masses are of the following form, (8) (4) (4) (4) Wν = αN1 Λ(N1 N1 )1 Y1 +αN2 Λ(N2 N3 )1 Y1 +αN3 Λ(N3 N3 )100 Y10 +αν1 (N1 F )3 Y3 H5 (2) (2) +αν2 (N2 F )3 Y3 H5 +αν3 (N3 F )3 Y3 H5 , (2) (4) (4) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (4) (4) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (6) (8) (8) (8) Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T2 )10 Y100 H5 +αu3 (T2 T3 )1 Y1 H5 +αu4 (T3 T3 )100 Y10 H5 . (4.58) The matrices MN and Y5ν are identical with those of the model V4 as well. The matrices Y5d and Y5u are the same as those of the model I17 as well. The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and αN2 , αN3 , αd0 2 , αd0 3 , αu1 , αu2 and αu4 are generically complex. • Model V8 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (10 , 10 , 1, 3, 100 , 1, 10 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (2, 4, 2, 0, 2, 2, 2) We can read out the superpotential for the fermion masses as, (4) (8) (4) (2) Wν = αN1 Λ(N1 N1 )100 Y10 +αN2 Λ(N2 N2 )100 Y10 +αN3 Λ(N3 N3 )1 Y1 +αν1 (N1 F )3 Y3 H5 (4) (2) +αν2 (N2 F )3 Y3 H5 +αν3 (N3 F )3 Y3 H5 , (2) (2) (2) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (2) (4) (4) (2) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (4) (4) Wu = αu1 (T1 T2 )100 Y10 H5 +αu2 (T1 T3 )1 Y1 H5 +αu3 (T2 T2 )1 Y1 H5 +αu4 (T3 T3 )100 Y10 H5 . (4.59) – 29 – JHEP04(2021)239 The matrices MN and Y5ν are identical with the corresponding ones of the model V4 as well. The Y5d matrix coincides with that of the model I16 . The Y5u matrix is identical with that of the model I12 . The phases of the parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be removed by field redefinition, and the remaining parameters are complex. We can straightforwardly read out the right-handed neutrino mass matrix and the Yukawa matrices as follows,  mνM (4) α Y 0  N1 1  = Λ 0 0  (2) 0 (8) α N2 Y 1 0 0 (2) αν1 Y3,2 (4) αν2 Y3,2 (2) αν3 Y3,3 (2)  αν1 Y3,1 (4)  αν2 Y3,1  . (2) αν3 Y3,2 (4.60) The matrices Y5d and Y5u are identical with the corresponding ones of the model V4 as well. The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and αN2 , αN3 , αd0 2 , αd0 3 , αu1 , αu2 and αu4 are generically complex. • Model V9 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (10 , 10 , 1, 3, 100 , 10 , 10 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (2, 4, 2, 0, 2, 2, 4) The superpotentials relevant to quark and lepton masses are of the form, (4) (8) (4) (2) Wν = αN1 Λ(N1 N1 )100 Y10 +αN2 Λ(N2 N2 )100 Y10 +αN3 Λ(N3 N3 )1 Y1 +αν1 (N1 F )3 Y3 H5 (4) (2) +αν2 (N2 F )3 Y3 H5 +αν3 (N3 F )3 Y3 H5 , (2) (2) (4) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (2) (4) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (4) (6) (4) (8) Wu = αu1 (T1 T2 )1 Y1 H5 +αu2 (T1 T3 )1 Y1 H5 +αu3 (T2 T2 )100 Y10 H5 +αu4 (T3 T3 )100 Y10 H5 . (4.61) The matrices MN and Y5ν are identical with the corresponding ones of the model V8 as well. The Y5d matrix is the same as that of the model I15 . The Y5u matrix is identical with the corresponding one of the model I11 . The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and αN2 , αN3 , αd0 2 , αd0 3 , αu1 , αu2 and αu4 are complex. • Model V10 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (10 , 10 , 1, 3, 100 , 100 , 10 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (2, 4, 2, 0, 2, 4, 2) We can read out the superpotential for quark and leptons as follows, (4) (8) (4) (2) Wν = αN1 Λ(N1 N1 )100 Y10 +αN2 Λ(N2 N2 )100 Y10 +αN3 Λ(N3 N3 )1 Y1 +αν1 (N1 F )3 Y3 H5 (4) (2) +αν2 (N2 F )3 Y3 H5 +αν3 (N3 F )3 Y3 H5 , (2) (4) (2) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (4) (2) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (4) (8) (6) (4) Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T2 )10 Y100 H5 +αu3 (T2 T3 )1 Y1 H5 +αu4 (T3 T3 )100 Y10 H5 . (4.62) – 30 – JHEP04(2021)239 αν Y  1 3,3 (4) ν Y5 =   αν2 Y3,3 (2) αν3 Y3,1  0  , 0  (4) α N3 Y 1 The matrices MN and Y5ν are the same as those of the model V8 as well. The Y5d matrix coincides with the corresponding one of the model I16 . The Y5u matrix is in common with that of the model I12 . The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can taken to be real without loss of generality, while the other parameters are complex. • Model V11 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (10 , 10 , 1, 3, 100 , 100 , 10 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (2, 4, 2, 0, 2, 4, 4) We can read out the superpotential for quark and leptons as follows, (4) (8) (4) (2) Wν = αN1 Λ(N1 N1 )100 Y10 +αN2 Λ(N2 N2 )100 Y10 +αN3 Λ(N3 N3 )1 Y1 +αν1 (N1 F )3 Y3 H5 (2) (2) (4) (4) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (4) (4) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (6) (8) (8) (8) Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T2 )10 Y100 H5 +αu3 (T2 T3 )1 Y1 H5 +αu4 (T3 T3 )100 Y10 H5 . (4.63) The matrices MN and Y5ν are identical with the corresponding ones of the model V8 as well. The matrices Y5d and Y5u are identical with the corresponding ones of the model I17 as well. The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and the remaining parameters are complex. • Model V12 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (100 , 100 , 1, 3, 100 , 1, 10 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (2, 4, 2, 0, 2, 2, 2) We can read out the superpotential for the fermion masses as, (4) (8) (4) (2) Wν = αN1 Λ(N1 N3 )100 Y10 +αN2 Λ(N2 N2 )10 Y100 +αN3 Λ(N3 N3 )1 Y1 +αν1 (N1 F )3 Y3 H5 (4) (2) +αν2 (N2 F )3 Y3 H5 +αν3 (N3 F )3 Y3 H5 , (2) (2) (2) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (2) (4) (4) (2) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (4) (4) Wu = αu1 (T1 T2 )100 Y10 H5 +αu2 (T1 T3 )1 Y1 H5 +αu3 (T2 T2 )1 Y1 H5 +αu4 (T3 T3 )100 Y10 H5 , (4.64) which lead to  mνM 0   = Λ 0 (4) α N1 Y 1 0  (2) αν Y  1 3,2 (4) ν  Y5 =  αν2 Y3,2 (2) αν3 Y3,1 0 (8) αN2 Y100 0 (2) αν1 Y3,1 (4) αν2 Y3,1 (2) αν3 Y3,3 (4)  α N1 Y 1 0  , 0  (4) α N3 Y 1 (2)  αν1 Y3,3 (4)  αν2 Y3,3  . (2) αν3 Y3,2 (4.65) The matrices Y5d and Y5u are in common with the corresponding ones of the model V4 as well. The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can taken to be real without loss of generality, while the remaining parameters are complex. – 31 – JHEP04(2021)239 (4) +αν2 (N2 F )3 Y3 H5 +αν3 (N3 F )3 Y3 H5 , • Model V13 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (100 , 100 , 1, 3, 100 , 1, 10 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (2, 4, 2, 0, 2, 4, 4) We can read out the superpotential for the fermion masses as, (4) (8) (4) (2) Wν = αN1 Λ(N1 N3 )100 Y10 +αN2 Λ(N2 N2 )10 Y100 +αN3 Λ(N3 N3 )1 Y1 +αν1 (N1 F )3 Y3 H5 (4) (2) +αν2 (N2 F )3 Y3 H5 +αν3 (N3 F )3 Y3 H5 , (2) (4) (4) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (4) (4) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (8) (8) (8) The matrices MN and Y5ν are the same as those of the model V12 as well. The matrices Y5d and Y5u are identical with the corresponding ones of the model I14 as well. The phases of the parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be removed by field redefinition, and the remaining parameters are complex. • Model V14 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (100 , 100 , 1, 3, 100 , 1, 1) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (2, 4, 2, 0, 2, 2, 4) The modular invariant superpotential for quark and lepton mass is given by (4) (8) (4) (2) Wν = αN1 Λ(N1 N3 )100 Y10 +αN2 Λ(N2 N2 )10 Y100 +αN3 Λ(N3 N3 )1 Y1 +αν1 (N1 F )3 Y3 H5 (4) (2) +αν2 (N2 F )3 Y3 H5 +αν3 (N3 F )3 Y3 H5 , (2) (2) (4) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3 H5 (2) (2) (4) (4) (4) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3 H45 , (6) (8) Wu = αu1 (T1 T2 )100 Y10 H5 +αu2 (T2 T2 )1 Y1 H5 +αu3 (T2 T3 )1 Y1 H5 +αu4 (T3 T3 )1 Y1 H5 . (4.67) The matrices MN and Y5ν are the same as those of the model V12 as well. The Y5d matrix is the same as the corresponding one of the model I1 . The Y5u matrix is identical with that of the model I6 . The phases of the parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 are unphysical, and the other parameters are complex. • Model V15 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 100 , 10 , 3, 10 , 100 , 100 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (0, 0, 0, 2, 0, 0, 4) The quark and lepton masses are described by the following superpotential: (2) (2) Wν = αN1 Λ(N1 N1 )1 +αN2 Λ(N2 N3 )1 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3 H5 (2) +αν3 (N3 F )3 Y3 H5 , (2) (2) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (2) (2) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (4) (8) Wu = αu1 (T1 T2 )1 H5 +αu2 (T1 T3 )1 Y1 H5 +αu3 (T3 T3 )10 Y100 H5 , – 32 – (4.68) JHEP04(2021)239 (6) Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T2 )1 Y1 H5 +αu3 (T2 T3 )10 Y100 H5 +αu4 (T3 T3 )100 Y10 H5 . (4.66) which lead to  mνM α N1  = Λ 0 0   0 0 α N2 0  α N2  , 0  (2) αν1 Y3,3 (2) αν2 Y3,1 (2) αν3 Y3,2 αd1 Y3,3 (2) αd2 Y3,2 (2) Y5d =   αd1 Y3,2 (2) αd1 Y3,1 αd2 Y3,1   0  Y5u =  α u1  (4) α u2 Y 1 αν1 Y3,2 (2)  αν2 Y3,3  , (2) αν3 Y3,1 (2) αd3 Y3I,2 + αd4 Y3II,2 (2) (6) (6) αd3 Y3I,1 + αd4 Y3II,1  , (2) αd2 Y3,3 (2)  (2) (6)   (6) (6) αd3 Y3I,3 + αd4 Y3II,3 (4) α u1 0 0 (6)  α u2 Y 1  . 0  (8) αu3 Y100 (4.69) The phases of the parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 are unphysical, and the remaining parameters are complex. • Model V16 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 100 , 10 , 3, 10 , 100 , 1) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (0, 0, 0, 2, 0, 0, 4) The quark and lepton masses are described by the following superpotential: (2) (2) Wν = αN1 Λ(N1 N1 )1 +αN2 Λ(N2 N3 )1 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3 H5 (2) +αν3 (N3 F )3 Y3 H5 , (2) (2) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (2) (2) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (4) (8) Wu = αu1 (T1 T2 )1 H5 +αu2 (T2 T3 )100 Y10 H5 +αu3 (T3 T3 )1 Y1 H5 . (4.70) The right-handed neutrino mass matrix and the Yukawa matrix are given by αd1 Y3,3 (2) αd2 Y3,2 (2) Y5d =   αd1 Y3,2 (2) αd1 Y3,1 αd2 Y3,1    0  u  Y 5 =  α u1 0 (2) αd3 Y3I,1 + αd4 Y3II,1 (2) (6) (6) αd3 Y3I,3 + αd4 Y3II,3  , (2) αd2 Y3,3 α u1 0 (4) α u2 Y 1 0 (6) (6)   (6) (6) αd3 Y3I,2 + αd4 Y3II,2  0 (4)  α u2 Y 1 0  . (8) α u3 Y 1 (4.71) The matrices MN and Y5ν are identical with the corresponding ones of the model V15 as well. The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and the other parameters are complex. – 33 – JHEP04(2021)239 αν Y  1 3,1 (2) ν  Y5 =  αν2 Y3,2 (2) αν3 Y3,3 • Model V17 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 100 , 10 , 3, 10 , 100 , 1) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (0, 0, 0, 4, 0, 0, 4) We can read out the superpotentials relevant to quark and lepton masses (4) (4) Wν = αN1 Λ(N1 N1 )1 +αN2 Λ(N2 N3 )1 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3 H5 (4) +αν3 (N3 F )3 Y3 H5 , (4) (4) (8) (8) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (4) (4) (8) (8) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (8) (4.72) The right-handed neutrino mass matrix and the Yukawa matrix are given by   αν Y  1 3,1 (4) ν  Y5 =  αν2 Y3,2 (4) αν3 Y3,3 (4) αν1 Y3,3 (4) αν2 Y3,1 (4) αν3 Y3,2 αd1 Y3,3 (4) αd2 Y3,2 (4) Y5d =   αd1 Y3,2 (4) αd1 Y3,1 αd2 Y3,1  αν1 Y3,2 (4)  αν2 Y3,3  , (4) αν3 Y3,1 (4) αd3 Y3I,1 + αd4 Y3II,1 (4) (8) (8) αd3 Y3I,3 + αd4 Y3II,3  . (4) αd2 Y3,3 (4)  (4) (8) (8)  (4.73)  (8) αd3 Y3I,2 + (8) αd4 Y3II,2 The matrices MN and Y5u are identical with those of the models V15 and V16 respectively. The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can taken to be real without loss of generality, while the remaining parameters αN2 , αd4 , αd0 2 , αd0 3 , αd0 4 , αu1 and αu2 are complex. • Model V18 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 100 , 10 , 3, 10 , 100 , 1) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (2, 0, 0, 2, 0, 0, 4) The quark and lepton masses are described by the following superpotential: (4) (4) (2) Wν = αN1 Λ(N1 N1 )1 Y1 +αN2 Λ(N2 N3 )1 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3 H5 (2) +αν3 (N3 F )3 Y3 H5 , (2) (2) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (2) (2) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (4) (8) Wu = αu1 (T1 T2 )1 H5 +αu2 (T2 T3 )100 Y10 H5 +αu3 (T3 T3 )1 Y1 H5 , which gives rise to  mνM (4) α N1 Y 1  = Λ 0 0 0 0 α N2 0  α N2  . 0 (4.74)  (4.75) The Y5ν matrix is identical with the corresponding one of the model V4 . The matrices Y5d and Y5u are identical with those of the model V16 as well. The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and the other parameters are complex. – 34 – JHEP04(2021)239 (4) Wu = αu1 (T1 T2 )1 H5 +αu2 (T2 T3 )100 Y10 H5 +αu3 (T3 T3 )1 Y1 H5 . • Model V19 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 100 , 10 , 3, 10 , 100 , 100 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (0, 0, 0, 4, 0, 0, 4) The modular invariant superpotential for quark and lepton mass is given by (4) (4) Wν = αN1 Λ(N1 N1 )1 +αN2 Λ(N2 N3 )1 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3 H5 (4) +αν3 (N3 F )3 Y3 H5 , (4) (4) (8) (8) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (4) (8) (8) (4) (8) Wu = αu1 (T1 T2 )1 H5 +αu2 (T1 T3 )1 Y1 H5 +αu3 (T3 T3 )10 Y100 H5 . (4.76) The right-handed neutrino mass matrix and the Yukawa matrix are given by αd1 Y3,3 (4) αd2 Y3,2 (4) Y5d =   αd1 Y3,2 (4) αd1 Y3,1 αd2 Y3,1   (4) αd3 Y3I,2 + αd4 Y3II,2 (4) (8) (8) αd3 Y3I,1 + αd4 Y3II,1  . (4) αd2 Y3,3 (8) (8)  (4.77)  (8) αd3 Y3I,3 + (8) αd4 Y3II,3 The matrices MN and Y5u are identical with the corresponding ones of the model V15 as well. The Y5ν matrix is identical with that of the model V17 . The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can taken to be real without loss of generality, while the other parameters are complex. • Model V20 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 100 , 10 , 3, 10 , 100 , 100 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (2, 0, 0, 2, 0, 0, 4) The modular invariant superpotential is of the following form: (4) (4) (2) Wν = αN1 Λ(N1 N1 )1 Y1 +αN2 Λ(N2 N3 )1 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3 H5 (2) +αν3 (N3 F )3 Y3 H5 , (2) (2) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (2) (2) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (4) (8) Wu = αu1 (T1 T2 )1 H5 +αu2 (T1 T3 )1 Y1 H5 +αu3 (T3 T3 )10 Y100 H5 . (4.78) The MN matrix coincides with that of the model V18 . The Y5ν matrix is the same as that of the model V4 . The matrices Y5d and Y5u are identical with those of the model V15 as well. The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and αN2 , αd4 , αd0 2 , αd0 3 , αd0 4 , αu1 and αu2 are complex. – 35 – JHEP04(2021)239 (4) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , • Model V21 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 100 , 10 , 3, 100 , 10 , 10 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (0, 0, 0, 2, 2, 0, 4) The modular invariant superpotential is of the following form: (2) (2) Wν = αN1 Λ(N1 N1 )1 +αN2 Λ(N2 N3 )1 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3 H5 (2) +αν3 (N3 F )3 Y3 H5 , (4) (2) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (4) (2) (6) (4) (8) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (4.79) The matrices MN and Y5ν are identical with the corresponding ones of the model V15 as well. The matrices Y5d and Y5u are the same as those of the model I2 as well. The phases of the parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 are unphysical, and the remaining parameters αN2 , αd4 , αd0 2 , αd0 3 , αd0 4 , αu1 and αu2 are complex. • Model V22 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 100 , 10 , 3, 100 , 10 , 10 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (2, 0, 0, 2, 2, 0, 4) The modular invariant superpotential for quark and lepton mass is given by (4) (4) (2) Wν = αN1 Λ(N1 N1 )1 Y1 +αN2 Λ(N2 N3 )1 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3 H5 (2) +αν3 (N3 F )3 Y3 H5 , (4) (2) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (4) (2) (6) (4) (8) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (6) Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T3 )100 Y10 H5 +αu3 (T3 T3 )100 Y10 H5 . (4.80) The MN matrix is identical with the corresponding one of the model V18 . The matrix d u is the same as that of the model V4 . The matrices Y5 and Y5 are identical with those of the model I2 as well. The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and the remaining parameters αN2 , αd4 , αd0 2 , αd0 3 , αd0 4 , αu1 and αu2 are complex. Y5ν • Model V23 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 100 , 10 , 3, 100 , 100 , 10 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (0, 0, 0, 2, 0, 2, 4) The modular invariant superpotentials for quark and lepton masses are of the following form, (2) (2) Wν = αN1 Λ(N1 N1 )1 +αN2 Λ(N2 N3 )1 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3 H5 (2) +αν3 (N3 F )3 Y3 H5 , (2) (4) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (2) (4) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (4) (6) (8) Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T3 )1 Y1 H5 +αu3 (T3 T3 )100 Y10 H5 . – 36 – (4.81) JHEP04(2021)239 (6) Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T3 )100 Y10 H5 +αu3 (T3 T3 )100 Y10 H5 . The matrices MN and Y5ν are identical with those of the model V15 as well. The matrices Y5d and Y5u are identical with those of the model I3 as well. The phases of the parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be removed by field redefinition, and the remaining parameters are complex. • Model V24 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 100 , 10 , 3, 100 , 100 , 10 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (2, 0, 0, 2, 0, 2, 4) The superpotentials relevant to quark and lepton masses are of the form, (4) (4) (2) (2) +αν3 (N3 F )3 Y3 H5 , (2) (4) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (2) (4) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (4) (6) (8) Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T3 )1 Y1 H5 +αu3 (T3 T3 )100 Y10 H5 . (4.82) The MN matrix is identical with that of the model V18 . The Y5ν matrix coincides with that of the model V4 . The matrices Y5d and Y5u are identical with those of the model I3 as well. The phases of the parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be removed by field redefinition, and the other parameters are complex. • Model V25 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (10 , 10 , 100 , 3, 10 , 100 , 1) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (0, 2, 0, 2, 0, 0, 4) The modular invariant superpotentials for quark and lepton masses are of the following form, (4) (2) (4) Wν = αN1 Λ(N1 N3 )1 +αN2 Λ(N2 N2 )100 Y10 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3 H5 (2) +αν3 (N3 F )3 Y3 H5 , (2) (2) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (2) (2) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (4) (8) Wu = αu1 (T1 T2 )1 H5 +αu2 (T2 T3 )100 Y10 H5 +αu3 (T3 T3 )1 Y1 H5 . (4.83) The right-handed neutrino mass matrix and the Yukawa matrix are given by 0  = Λ 0 α N1 0 (4) α N2 Y 1 0 0  mνM  (2) αν Y  1 3,3 (4) Y5ν =   αν2 Y3,3 (2) αν3 Y3,2 (2) αν1 Y3,2 (4) αν2 Y3,2 (2) αν3 Y3,1  α N1  0 , 0 (2)  αν1 Y3,1 (4)  αν2 Y3,1  . (2) αν3 Y3,3 (4.84) The matrices Y5d and Y5u are identical with those of the model V16 as well. The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and αN2 , αd4 , αd0 2 , αd0 3 , αd0 4 , αu1 and αu2 are generically complex. – 37 – JHEP04(2021)239 Wν = αN1 Λ(N1 N1 )1 Y1 +αN2 Λ(N2 N3 )1 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3 H5 • Model V26 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (10 , 10 , 100 , 3, 10 , 100 , 100 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (0, 2, 0, 2, 0, 0, 4) The modular invariant superpotentials for quark and lepton masses are of the following form, (4) (2) (4) Wν = αN1 Λ(N1 N3 )1 +αN2 Λ(N2 N2 )100 Y10 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3 H5 (2) +αν3 (N3 F )3 Y3 H5 , (2) (2) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (2) (6) (6) (4) (8) Wu = αu1 (T1 T2 )1 H5 +αu2 (T1 T3 )1 Y1 H5 +αu3 (T3 T3 )10 Y100 H5 . (4.85) The matrices MN and Y5ν are identical with those of the model V25 as well. The matrices Y5d and Y5u are identical with those of the model V15 as well. The parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be taken real by field redefinition, and αN2 , αd4 , αd0 2 , αd0 3 , αd0 4 , αu1 and αu2 are generically complex. • Model V27 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (10 , 10 , 100 , 3, 100 , 10 , 10 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (0, 2, 0, 2, 2, 0, 4) We can read out the superpotential for the fermion masses as, (4) (2) (4) Wν = αN1 Λ(N1 N3 )1 +αN2 Λ(N2 N2 )100 Y10 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3 H5 (2) +αν3 (N3 F )3 Y3 H5 , (4) (2) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (4) (2) (6) (4) (8) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (6) Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T3 )100 Y10 H5 +αu3 (T3 T3 )100 Y10 H5 . (4.86) The matrices MN and Y5ν are identical with those of the model V25 as well. The matrices Y5d and Y5u are the same as those of the model I2 as well. The phases of the parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 are unphysical, and the remaining parameters αN2 , αd4 , αd0 2 , αd0 3 , αd0 4 , αu1 and αu2 are complex. • Model V28 : (ρN1 , ρN2 , ρN3 , ρF , ρT1 , ρT2 , ρT3 ) = (10 , 10 , 100 , 3, 100 , 100 , 10 ) , (kN1 , kN2 , kN3 , kF , kT1 , kT2 , kT3 ) = (0, 2, 0, 2, 0, 2, 4) The quark and lepton masses are described by the following superpotential: (4) (2) (4) Wν = αN1 Λ(N1 N3 )1 +αN2 Λ(N2 N2 )100 Y10 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3 H5 (2) +αν3 (N3 F )3 Y3 H5 , (2) (4) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (2) (4) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (4) (6) (8) Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T3 )1 Y1 H5 +αu3 (T3 T3 )100 Y10 H5 . – 38 – (4.87) JHEP04(2021)239 (2) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , The matrices MN and Y5ν are identical with those of the model V25 as well. The matrices Y5d and Y5u are identical with those of the model I3 as well. The phases of the parameters αN1 , αν1 , αν2 , αν3 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be removed by field redefinition, and αN2 , αd4 , αd0 2 , αd0 3 , αd0 4 , αu1 and αu2 are complex. 4.4 Type-VII • Model VII 1 : (ρN1 , ρN2 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 100 , 3, 100 , 1, 1) , (kN1 , kN2 , kF , kT1 , kT2 , kT3 ) = (1, 3, 3, 1, 1, 3) (4) (4) (6) (6) Wν = αN1 Λ(N1 N2 )100 Y10 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3,I H5 +αν3 (N2 F )3 Y3,II H5 , (4) (4) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (4) (4) (4) (4) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (6) Wu = αu1 (T1 T3 )100 Y10 H5 +αu2 (T2 T3 )1 Y1 H5 +αu3 (T3 T3 )1 Y1 H5 . (4.88) which gives rise to mνM =Λ (4) 0 (4) α N1 Y 1 0 α N1 Y 1 0 0 (4) Y5ν = αν1 Y3,1 (6) (6) αν2 Y3I,2 + αν3 Y3II,2 ! , (4) αν1 Y3,3 (6) (6) αν2 Y3I,1 + αν3 Y3II,1 (4) αν1 Y3,2 (6) (6) αν2 Y3I,3 + αν3 Y3II,3 ! . (4.89) The matrices Y5d and Y5u are in common with the corresponding ones of the models I6 and I1 respectively. The parameters αN1 , αν1 , αν2 , αd1 , αd2 , αd3 , αd0 1 and αu3 can taken to be real without loss of generality, while αν3 , αd4 , αd0 2 , αd0 3 , αd0 4 , αu1 and αu2 are complex. • Model VII 2 : (ρN1 , ρN2 , ρF , ρT1 , ρT2 , ρT3 ) = (10 , 10 , 3, 100 , 1, 1) , (kN1 , kN2 , kF , kT1 , kT2 , kT3 ) = (1, 3, 3, 1, 1, 3) We can read out the superpotential for quark and leptons as follows, (4) (4) (6) (6) Wν = αN1 Λ(N1 N2 )100 Y10 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3,I H5 +αν3 (N2 F )3 Y3,II H5 , (4) (4) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (4) (4) (4) (4) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (6) Wu = αu1 (T1 T3 )100 Y10 H5 +αu2 (T2 T3 )1 Y1 H5 +αu3 (T3 T3 )1 Y1 H5 . (4.90) We can straightforwardly read out the right-handed neutrino mass matrix and the Yukawa matrices as follows, (4) Y5ν = αν1 Y3,3 (6) (6) αν2 Y3I,3 + αν3 Y3II,3 (4) αν1 Y3,2 (6) (6) αν2 Y3I,2 + αν3 Y3II,2 – 39 – (4) αν1 Y3,1 (6) (6) αν2 Y3I,1 + αν3 Y3II,1 ! . (4.91) JHEP04(2021)239 The modular invariant superpotentials for quark and lepton masses are of the following form, The MN matrix is in common with that of the model VII 1 . The Y5d matrix is the same as that of the model I6 . The Y5u matrix is in common with that of the model I1 . The phases of the parameters αN1 , αν1 , αν2 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be removed by field redefinition, and αν3 , αd4 , αd0 2 , αd0 3 , αd0 4 , αu1 and αu2 are generically complex. • Model VII 3 : (ρN1 , ρN2 , ρF , ρT1 , ρT2 , ρT3 ) = (10 , 100 , 3, 100 , 1, 1) , (kN1 , kN2 , kF , kT1 , kT2 , kT3 ) = (1, 3, 3, 1, 1, 3) We can read out the superpotential for quark and leptons as follows, (4) (4) (6) (6) (4) (4) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (4) (4) (4) (4) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (6) Wu = αu1 (T1 T3 )100 Y10 H5 +αu2 (T2 T3 )1 Y1 H5 +αu3 (T3 T3 )1 Y1 H5 , (4.92) which lead to mνM =Λ 0 (4) α N1 Y 1 (4) α N1 Y 1 0 (4) Y5ν = αν1 Y3,3 (6) (6) αν2 Y3I,2 + αν3 Y3II,2 ! , (4) αν1 Y3,2 (6) (6) αν2 Y3I,1 + αν3 Y3II,1 (4) αν1 Y3,1 (6) (6) αν2 Y3I,3 + αν3 Y3II,3 ! . (4.93) The matrices Y5d and Y5u coincide with those of the model VII 1 as well. The phases of the parameters αN1 , αν1 , αν2 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be removed by field redefinition, and αν3 , αd4 , αd0 2 , αd0 3 , αd0 4 , αu1 and αu2 are generically complex. • Model VII 4 : (ρN1 , ρN2 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 1, 3, 100 , 100 , 10 ) , (kN1 , kN2 , kF , kT1 , kT2 , kT3 ) = (0, 2, 2, 0, 2, 4) We can read out the superpotential for quark and leptons as follows, (4) (2) (4) Wν = αN1 Λ(N1 N1 )1 +αN2 Λ(N2 N2 )1 Y1 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3 H5 , (2) (4) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (2) (4) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (4) (6) (8) Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T3 )1 Y1 H5 +αu3 (T3 T3 )100 Y10 H5 . (4.94) The right-handed neutrino mass matrix and the Yukawa coupling matrices are predicted to be mνM =Λ 0 (4) α N2 Y 1 α N1 0 (2) Y5ν = αν1 Y3,1 (4) αν2 Y3,1 ! (2) αν1 Y3,3 (4) αν2 Y3,3 , (2) αν1 Y3,2 (4) αν2 Y3,2 ! . (4.95) The matrices Y5d and Y5u are the same as those of the model I3 as well. The phases of the parameters αN1 , αν1 , αν2 , αd1 , αd2 , αd3 , αd0 1 and αu3 are unphysical, and the other parameters are complex. – 40 – JHEP04(2021)239 Wν = αN1 Λ(N1 N2 )1 Y1 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3,I H5 +αν3 (N2 F )3 Y3,II H5 , • Model VII 5 : (ρN1 , ρN2 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 10 , 3, 100 , 100 , 10 ) , (kN1 , kN2 , kF , kT1 , kT2 , kT3 ) = (0, 2, 2, 0, 2, 4) We can read out the superpotentials relevant to quark and lepton masses (4) (2) (4) Wν = αN1 Λ(N1 N1 )1 +αN2 Λ(N2 N2 )100 Y10 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3 H5 , (2) (4) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (2) (4) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (4) (6) (8) (4.96) The right-handed neutrino mass matrix and the Yukawa matrix are given by 0 (4) α N2 Y 1 0 mνM = Λ α N1 0 Y5ν = αν1 Y3,1 (4) αν2 Y3,3 (2) ! (2) αν1 Y3,3 (4) αν2 Y3,2 , (2) αν1 Y3,2 (4) αν2 Y3,1 ! (4.97) . The matrices Y5d and Y5u coincide with those of the model VII 4 as well. The parameters αN1 , αν1 , αν2 , αd1 , αd2 , αd3 , αd0 1 and αu3 are real, and αN2 , αd4 , αd0 2 , αd0 3 , αd0 4 , αu1 and αu2 are generically complex. • Model VII 6 : (ρN1 , ρN2 , ρF , ρT1 , ρT2 , ρT3 ) = (1, 10 , 3, 100 , 100 , 10 ) , (kN1 , kN2 , kF , kT1 , kT2 , kT3 ) = (2, 2, 2, 0, 2, 4) We can read out the superpotentials relevant to quark and lepton masses (4) (4) (4) (4) Wν = αN1 Λ(N1 N1 )1 Y1 +αN2 Λ(N2 N2 )100 Y10 +αν1 (N1 F )3 Y3 H5 +αν2 (N2 F )3 Y3 H5 , (2) (4) (6) (6) Wd = αd1 (F T1 )3 Y3 H5 +αd2 (F T2 )3 Y3 H5 +αd3 (F T3 )3 Y3,I H5 +αd4 (F T3 )3 Y3,II H5 (2) (4) (6) (6) +αd0 1 (F T1 )3 Y3 H45 +αd0 2 (F T2 )3 Y3 H45 +αd0 3 (F T3 )3 Y3,I H45 +αd0 4 (F T3 )3 Y3,II H45 , (4) (6) (8) Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T3 )1 Y1 H5 +αu3 (T3 T3 )100 Y10 H5 , (4.98) which gives rise to (4) mνM =Λ α N1 Y 1 0 (4) Y5ν = αν1 Y3,1 (4) αν2 Y3,3 0 (4) α N2 Y 1 0 (4) αν1 Y3,3 (4) αν2 Y3,2 ! , (4) αν1 Y3,2 (4) αν2 Y3,1 ! . (4.99) The matrices Y5d and Y5u are in common with the corresponding ones of the model VII 4 as well. The phases of the parameters αN1 , αν1 , αν2 , αd1 , αd2 , αd3 , αd0 1 and αu3 can be removed by field redefinition, and the other parameters are complex. – 41 – JHEP04(2021)239 Wu = αu1 (T1 T3 )1 Y1 H5 +αu2 (T2 T3 )1 Y1 H5 +αu3 (T3 T3 )100 Y10 H5 . 5 Numerical results χ2total = X i ξi − ξi σi !2 , (5.1) where ξi and σi refer to the global best fit values and the 1σ deviations of the corresponding observables and their values are listed in table 3. The total χ2total can be split into the lepton and quark contributions χ2l and χ2q respectively with χ2total = χ2l +χ2q . We construct the lepton sector χ2l function with the mass ratios l , sin2 θ l , sin2 θ l , me /mµ , mµ /mτ , ∆m221 /∆m231 and the lepton mixing parameters sin2 θ12 13 23 l . The quark sector χ2 function is constructed from the quark mass ratios m /m , δCP u c q q q q q mc /mt , md /ms , ms /mb and the quark mixing parameters θ12 , θ13 , θ23 , δCP . The tau mass mτ is fixed to its experimental best fit value to determine the common overall scale of the charged lepton and down quark mass matrices, the contribution from the mass ratio mb /mτ is included into the χ2q . Notice that the overall scale factors of the mass matrices don’t affect the value of χ2total . The values of the quark and charged lepton masses and the CKM mixing parameters at the GUT scale are taken from ref. [73] with tan β = 10 and the SUSY breaking scale MSUSY = 10 TeV. The experimental values of the neutrino mixing parameters are taken from NuFIT v5.0 with Super-Kamiokanda atmospheric data [4]. Since the normal ordering (NO) neutrino mass spectrum is slightly preferred over the inverted ordering (IO) masses by the present data [4], we focus on the NO in the following. For any given values of the input parameters of a model, we diagonalize the mass matrices to extract the lepton and quark masses and mixing matrices, and then the value of χ2total is calculated. The absolute value of all dimensionless parameters are treated as random numbers varying freely between 0 and 108 and their phases are distributed uniformly in then region [0, 2π], while the VEV ofothe modulus τ is limited in the fundamental region D = τ |Im(τ ) > 0, |Re(τ )| ≤ 12 , |τ | ≥ 1 . We numerically search for the global minimum of χ2total by using the minimization algorithms incorporated in the CERN developed package – 42 – JHEP04(2021)239 We have numerically scanned the parameter space of each model to optimize the agreement between predictions and experimental data. The dynamics determining the VEV of the complex modulus τ is an open question now, consequently we treat the VEV of τ as a free parameter to match the experimental data. As explicitly shown in previous section, the phases of some coupling constants can be removed by a field redefinition while others are generally complex. Each model depends on a set of dimensionless input parameters and three overall mass scales for the up type quark, charged lepton and neutrino mass matrices. The dimensionless input parameters include the modulus τ and the ratios of the coupling constants and they determine the mixing angles, CP violation phases and the fermion mass ratios. As can be seen from eq. (3.17), the down-type quark and charged lepton mass matrices are closely related in SU(5) and they share a same overall factor. We can use the measured values of ∆m221 , mτ and mt to determine the overall mass parameters of the neutrino, the charged lepton, the up quark mass matrices. As a measure of goodness of fit, we use a χ2 function defined in the usual way, Best fit values and 1σ ranges mu /mc (1.9286 ± 0.6017) × 10−3 mc /mt (2.8213 ± 0.1195) × 10−3 md /ms (5.0523 ± 0.6191) × 10−2 ms /mb (1.8241 ± 0.1005) × 10−2 mb /mτ (0.7434 ± 0.0090) × 10−2 q δCP 69.213◦ ± 3.115◦ q θ12 0.22736 ± 0.00073 q θ13 q θ23 0.00349 ± 0.00013 me /mµ (4.73689 ± 0.04019) × 10−3 mµ /mτ (5.85684 ± 0.04654) × 10−2 l sin2 θ12 0.304+0.012 −0.012 l sin2 θ23 0.573+0.016 −0.020 l sin2 θ13 0.02219+0.00062 −0.00063 l /◦ δCP 197+27 −24 ∆m221 10−5 eV2 7.42+0.21 −0.20 ∆m231 10−3 eV2 2.517+0.026 −0.028 mt 87.4555 ± 2.0893 GeV mτ 1.30234 ± 0.0068 GeV 0.04015 ± 0.00064 Table 3. The best fit values and 1σ errors of the mass ratios and mixing parameters of quarks and leptons. The values of the quark and charged lepton masses and the CKM parameters at the GUT scale are taken from ref. [73] with SUSY breaking scale MSUSY = 10 TeV and tan β = 10, η̄b = 0.09375 [73]. The lepton mixing parameters are taken from [4] for normal ordering neutrino masses. TMinuit to optimize the values of the input parameters [74]. We have scanned all models with number of parameters (including the real and imaginary part of τ ) less than 25. Requiring χ2total < 100, we find 17 type-I, 2 type-II, 28 type-V and 6 type-VII models are viable. The representations and weight assignments of the matter fields for these models are summarized in table 2. Moreover, we display the best fit values of the free parameters and the predictions for fermion masses and flavor mixing parameters as well as the minimal values of χ2l , χ2q and χ2total in tables 4–16. The results of leptonic PMNS parameters and the quark CKM parameters are extracted in the standard way as defined in the Particle Data Group (PDG). For instance, the PDG parametrization of the lepton PMNS mixing – 43 – JHEP04(2021)239 Parameters matrix is as follow,  cl12 cl13 l sl13 e−iδCP sl12 cl13 l l l l l l l iδ l l l l l iδ UPMNS =   −s12 c23 − c12 s13 s23 e CP c12 c23 − s12 s13 s23 e CP l l sl12 sl23 − cl12 sl13 cl23 eiδCP −c12 sl23 − sl12 sl13 cl23 eiδCP  cl13 sl23 cl13 cl23   Q,  (5.2) i l , sl = sinl θ , δ l with clij = cos θij ij CP is the Dirac CP violating phase, and Q = diag(1, e ij α21 2 , α31 2 mββ = m1 cos2 θ12 cos2 θ13 + m2 sin2 θ12 cos2 θ13 eiα21 + m3 sin2 θ13 ei(α31 −2δCP ) , (5.3) which reduces to mββ = m2 sin2 θ12 cos2 θ13 eiφ + m3 sin2 θ13 e−2iδCP , (5.4) for m1 = 0. The current most stringent limit is mββ < 61 meV from KamLAND-Zen [75]. From the predicted values of lepton mixing angles and neutrino masses, one can easily – 44 – JHEP04(2021)239 e ) is the Majorana phase matrix factor where α21 and α31 are the so-called Majorana CP phases. If the lightest neutrino is massless m1 = 0 or m3 = 0, α31 is unphysical and the only physical Majorana phase is φ = α21 . The quark CKM mixing matrix is parameterized similar to eq. (5.2) without the Majorana phase matrix Q. As can be seen from tables 4 to 16, there are 13 models I4 , I5 , I7 , I9 , I10 , I11 , I12 , I15 , V5 , V6 , V7 , V18 and V25 in good agreement with the experiment data at 1σ level, and 13 models I6 , I13 , I16 , I17 , V9 , V11 , V12 , V15 , V16 , V17 , V19 , V20 and V26 are compatible with data at 3σ level. All these models depend on 24 real parameters including the real and imaginary part of τ . In order accommodate the quark and lepton mass hierarchies, we see that hierarchical values of the coupling constants are necessary, and this is expected to be naturally explained by the weighton mechanism [30]. It is remarkable that the model I1 has only 18 real parameters including Re(τ ) and Im(τ ) and the predictions are in l and δ q qualitative agreement with the observations, although sin2 θ23 CP are slightly above the 3σ allowed regions and the charm quark mass mc is a bit smaller and the up quark mass is vanishing with mu = 0. In all the 22 parameters models I2 , I3 , V1 , V2 , VII 1 , VII 2 , VII 3 together with the models V21 , V22 , V23 , V24 , V27 , V28 , VII 4 , VII 5 and VII 6 , the top left 2 × 2 block of the Yukawa coupling matrix Y5u is zero such that we have mu = 0 which is compatible with the tiny despite non-vanishing up quark mass. Moreover, there are only two right-handed neutrinos in the type-VII models, and the lightest neutrino is massless. We plot the best fit value of the modulus τ in figure 1, it can be seen that τ tends to distribute around the boundary of the fundamental domain D, in particular some are clustered close to the self-dual point τ = i where S is unbroken. The value of the Dirac CP l phase δCP is still unknown, the current and upcoming long-baseline experiments will be l able to place important constraints. As can be seen from figure 2, the predictions for δCP mostly lie in the region of [π, 3π/2] and they could be tested at future experiments. The most sensitive probe to whether neutrinos are Dirac or Majorana states is the neutrinoless double beta decay (0νββ): (A, Z) → (A, Z + 2) + e− + e− . The decay amplitude is proportional to the effective Majorana mass i l Figure 2. The predictions for the Dirac CP phase δCP . The left panel is for the models of type-I, type-II and type-VII, and the right is for the type-V models. determine the effective mass mββ . The predictions for mββ of the type-I, II and VII models are shown in figure 3 and those of type-V models are displayed in figure 4. There are many experiments which are in various stages of planning and construction. the new generation of 0νββ decay experiments will significantly increase the sensitivity to this rare process such that a considerable amount of our models are within the reach in foreseeable future. – 45 – JHEP04(2021)239 Figure 1. The best fit values of the complex modulus τ for the viable models summarized in table 2. 6 Conclusion GUTs are an important framework to understand the origin of the observed patterns of fermion masses and flavor mixing. The quark and leptons fields in each generation are embedded into a GUT multiplet so that they are closely related. It is usually assumed that the three generations of fermions are related by certain family symmetry to address the flavor mixing structure of quarks and leptons. The role of modular invariance as flavor symmetry can overcome the drawback of the conventional discrete flavor symmetry models. The modular symmetry is broken by the VEV of the complex modulus τ , and the Yukawa couplings are modular forms. Flavor models based on modular symmetry can be highly predictive, and the neutrino masses and lepton mixing can be predicted in terms of few – 46 – JHEP04(2021)239 Figure 3. The predictions for the effective mass of the neutrinoless double beta decay for the models of type I, type II and type VII. Notice that the lightest neutrino is massless for type VII models since only two right-handed neutrinos are introduced. The blue (red) lines denote the most general allowed regions for NO (IO) where the neutrino oscillation parameters are freely varied in their 3σ regions [4]. The vertical grey exclusion band denotes the bound on the lightest neutrino mass coming from the cosmological data Σi mi < 0.120 eV at 95% confidence level obtained by the Planck collaboration [2]. The current experimental bound from KamLAND-Zen [75] and the estimated experimental sensitivities of future 0νββ experiments are indicated by the horizontal lines. input parameters. Inspired by the success in the lepton sector, the modular symmetry has been extended to the quark sector [15, 18, 30, 32, 49, 50, 54]. In the present work, we imposed Γ3 ∼ = A4 modular symmetry on the SU(5) GUT to give a unified description of both quarks and leptons. In order to account for the mass difference of charged leptons and down-type quarks, we introduced the Higgs multiplet H45 besides H5 and H5 . All the three Higgs multiplets H5 , H5 and H45 were assumed to be invariant under A4 with zero modular weight. The neutrino masses are generated by the type-I seesaw mechanism, and both scenarios with three and two right-handed neutrinos were considered. The matter fields N , F and T transform as either triplets 3 or singlets 1, 10 , 100 under A4 . We have given the most general form of the quark and lepton mass matrices for different possible representation assignments, as shown in section 3. All possible models were classified according to the transformation properties of the matter fields, and we found there are five types of models for three right-handed neutrinos and two types of models for two right-handed neutrinos. If N , F and T are all singlets of A4 , the Yukawa superpotential would be less restricted by modular symmetry and generally more free parameters would be involved, hence we did not consider these cases in the present work since we were interested in the simplest possibilities. – 47 – JHEP04(2021)239 Figure 4. The predictions for the effective mass of the neutrinoless double beta decay for the type V models. We adopt the same convention as figure 3. Acknowledgments PC and GJD are grateful to Dr. Chang-Yuan Yao for his kind help on numerical analysis. PC is supported by the National Natural Science Foundation of China under Grant Nos 11847240. GJD is supported by the National Natural Science Foundation of China under Grant Nos 11975224, 11835013, 11947301, 12047502. SFK acknowledges the STFC Consolidated Grant ST/L000296/1 and the European Union’s Horizon 2020 Research and Innovation programme under Marie Skłodowska-Curie grant agreement HIDDeN European ITN project (H2020-MSCA-ITN-2019//860881-HIDDeN). A Results of the best fit for the benchmark models In this appendix, we collect the best fit values of the input parameters and the corresponding predictions for the quark and lepton masses and mixing parameters for the phenomenologically viable models listed in table 2. Here we have considered normal ordering neutrino masses which is slightly preferred by the experimental data [4]. – 48 – JHEP04(2021)239 We have performed a numerical scan over the parameter space of each model, and searched for the minimum of the χ2total function to optimize the agreement between predictions and data. For normal ordering neutrino masses, intensive numerical calculations revealed that there are 17 type-I models, 2 type-II models, 28 type-V and 6 type-VII models with χ2total < 100 with the number of real free parameters not larger than 24 including the real and imaginary part of τ . Out of the 54 considered models, we found that 12 type-I and 14 type-V models are in agreement with the experimental data at 3σ level, as can be seen from tables 4 to 16. All those phenomenological viable examples have 24 parameters. The predictions for the leptonic CP violating Dirac phase, the lightest neutrino mass and the neutrinoless double beta decay parameter as shown in the figures are observed to cover a wide range of possible values, but appear to be clustered around particular regions. Consequently the forthcoming generation of neutrino oscillation experiments sensitive to the leptonic CP phase, in conjunction with the upcoming neutrinoless double beta decay experiments, will together be able to discriminate between the various types of models based on Γ3 ∼ = A4 modular symmetry with SU(5) GUTs considered here. I1 I2 I3 Re(τ ) Im(τ ) |αν2 /αν1 | arg(αν2 /αν1 )/π αd1 /αd3 αd2 /αd3 |αd4 /αd3 | arg(αd4 /αd3 )/π αd0 1 /αd3 |αd0 2 /αd3 | arg(αd0 2 /αd3 )/π |αd0 3 /αd3 | arg(αd0 3 /αd3 )/π |αd0 4 /αd3 | arg(αd0 4 /αd3 )/π |αu1 /αu3 | arg(αu1 /αu3 )/π |αu2 /αu3 | arg(αu2 /αu3 )/π (αν21 vu2 /(αN1 Λ))/meV (αd3 vd )/GeV (αu3 vu )/GeV 0.4623 1.249 1.216 1.513 0.0275 0.465 — — −0.0296 0.482 0.979 0.333 2.677 × 10−4 — — 0.0143 0.973 0.0604 0.973 19.224 0.624 73.304 −0.2778 1.560 2.497 1.913 0.00253 2.089 × 10−6 2.731 1.300 0.00133 0.0534 0.203 0.616 1.690 0.506 0.307 0.00421 0.665 0.0514 0.639 7.806 0.556 191.031 −0.4732 1.574 1.961 1.634 0.0148 0.0844 7.592 0.678 0.00692 0.158 0.625 0.278 0.881 3.166 0.206 0.00574 1.636 0.0222 0.185 9.643 0.246 198.532 l sin2 θ13 2 l sin θ12 l sin2 θ23 l δCP /◦ α21 /◦ α31 /◦ 0.0214 0.312 0.631 127.565 233.130 247.772 0.0218 0.302 0.574 221.626 357.093 22.892 0.0222 0.306 0.567 198.745 172.063 171.828 m1 /meV m2 /meV m3 /meV mββ /meV 39.787 40.709 64.099 23.039 30.384 31.581 58.579 30.735 35.361 36.395 61.346 12.490 q θ12 q θ13 q θ23 q δCP /◦ 0.227 0.00349 0.0419 80.421 0.227 0.00349 0.0404 70.296 0.227 0.00349 0.0401 69.111 mu /mc mc /mt md /ms ms /mb 0.0 0.00223 0.0584 0.0195 0.0 0.00273 0.0528 0.0185 0.0 0.00284 0.0518 0.0182 mb /mτ 0.737 0.743 0.743 χ2l χ2q 2 χtotal 23.835 58.654 82.490 1.452 11.438 12.890 0.124 10.362 10.486 Table 4. The best fit values of the free parameters and the corresponding predictions for lepton and quark mixing parameters and fermion masses for the phenomenologically viable models of Type-I. – 49 – JHEP04(2021)239 Model I4 I5 I6 I7 Re(τ ) Im(τ ) |αν2 /αν1 | arg(αν2 /αν1 )/π αd1 /αd3 αd2 /αd3 |αd4 /αd3 | arg(αd4 /αd3 )/π αd0 1 /αd3 |αd0 2 /αd3 | arg(αd0 2 /αd3 )/π |αd0 3 /αd3 | arg(αd0 3 /αd3 )/π |αd0 4 /αd3 | arg(αd0 4 /αd3 )/π |αu1 /αu3 | arg(αu1 /αu3 )/π |αu2 /αu3 | arg(αu2 /αu3 )/π |αu4 /αu3 | arg(αu4 /αu3 )/π (αν21 vu2 /(αN1 Λ))/meV (αd3 vd )/GeV (αu3 vu )/GeV 0.1427 1.184 2.949 4.567 × 10−8 0.0523 0.00504 0.620 1.039 −0.0207 0.00137 0.0451 0.448 0.185 0.324 0.246 5.513 0.298 6.074 × 10−4 1.474 88.520 0.307 3.652 0.600 0.823 0.1601 1.188 2.937 0.00486 0.0839 0.00463 2.136 1.070 −0.0285 0.00134 1.956 0.607 0.148 0.245 0.577 3.942 0.0437 3.998 × 10−4 1.014 64.698 1.986 3.731 0.404 1.157 0.2830 1.429 2.609 0.0791 0.00416 0.0794 1.068 0.950 0.00114 0.0421 0.716 0.328 0.436 0.427 1.582 0.00415 0.647 6.784 × 10−4 0.174 19.236 0.128 6.333 0.831 4.587 −0.1956 1.103 3.051 1.999 0.220 0.0213 4.410 1.690 −0.0878 0.00866 1.991 1.735 0.688 1.263 1.465 5.555 0.238 6.807 × 10−4 1.460 82.789 1.915 3.180 0.136 0.746 l sin2 θ13 2 l sin θ12 l sin2 θ23 l δCP /◦ α21 /◦ α31 /◦ 0.0222 0.304 0.573 199.402 169.642 337.832 0.0222 0.304 0.573 197.516 193.611 25.864 0.0223 0.296 0.584 224.737 329.057 38.097 0.0222 0.304 0.574 198.558 169.259 337.628 m1 /meV m2 /meV m3 /meV mββ /meV 11.357 14.254 51.447 4.118 12.222 14.953 51.635 5.278 24.989 26.432 56.040 25.027 11.391 14.281 51.437 4.169 q θ12 q θ13 q θ23 q δCP /◦ 0.227 0.00350 0.0405 69.199 0.227 0.00349 0.0401 69.220 0.227 0.00348 0.0402 68.340 0.227 0.00349 0.0405 69.277 mu /mc mc /mt md /ms ms /mb 0.00195 0.00282 0.0506 0.0183 0.00193 0.00282 0.0505 0.0182 0.00215 0.00283 0.0499 0.0190 0.00198 0.00282 0.0507 0.0182 mb /mτ 0.744 0.743 0.743 0.743 2.071 0.808 2.879 0.00533 0.331 0.337 χ2l χ2q 2 χtotal 0.00882 0.233 0.242 −4 6.178 × 10 8.577 × 10−4 0.00148 Table 5. The best fit values of the free parameters and the corresponding predictions for lepton and quark mixing parameters and fermion masses for the phenomenologically viable models of Type-I (continuation of table 4). – 50 – JHEP04(2021)239 Model I8 I9 I10 I11 I12 Re(τ ) Im(τ ) |αN2 /αN1 | arg(αN2 /αN1 )/π |αN3 /αN1 | arg(αN3 /αN1 )/π |αν2 /αν1 | arg(αν2 /αν1 )/π αd1 /αd3 αd2 /αd3 |αd4 /αd3 | arg(αd4 /αd3 )/π αd0 1 /αd3 |αd0 2 /αd3 | arg(αd0 2 /αd3 )/π |αd0 3 /αd3 | arg(αd0 3 /αd3 )/π |αd0 4 /αd3 | arg(αd0 4 /αd3 )/π |αu1 /αu3 | arg(αu1 /αu3 )/π |αu2 /αu3 | arg(αu2 /αu3 )/π |αu4 /αu3 | arg(αu4 /αu3 )/π (αν21 vu2 /(αN1 Λ))/meV (αd3 vd )/GeV (αu3 vu )/GeV −0.03236 1.036 1.740 0.662 0.821 0.621 1.717 0.273 0.0349 0.00446 — — −0.0162 0.00107 0.139 0.0794 1.017 — — 10.616 0.273 1798.020 0.511 3.42 × 104 0.492 17.808 0.839 0.00188 −0.4738 1.637 — — — — 1.930 1.629 0.710 0.0297 11.704 0.458 −0.272 0.0104 0.0678 1.206 1.306 15.114 0.324 80.413 1.502 9332.920 0.766 4.625 × 105 1.418 10.479 0.0937 4.885 × 10−4 0.4264 1.125 — — — — 0.683 0.930 0.155 29.907 2.070 1.071 0.0455 15.226 1.570 0.430 1.768 0.883 0.204 0.00282 1.945 16.336 1.009 0.0855 0.900 11.659 0.0207 4.649 −0.4828 1.143 — — — — 2.273 1.624 0.0293 0.00281 0.148 0.980 −0.0117 9.652 × 10−4 0.0598 0.154 1.371 0.113 0.224 17.500 1.436 5227.610 1.804 1.491 × 105 1.974 5.633 1.104 6.475 × 10−4 0.3621 1.113 — — — — 3.224 0.0819 0.119 12.092 18.451 0.883 0.0542 23.972 0.00546 1.827 0.686 20.286 1.000 0.00311 0.322 15.062 0.675 0.0772 1.658 3.502 0.0179 4.204 l sin2 θ13 l sin2 θ12 l sin2 θ23 l δCP /◦ α21 /◦ α31 /◦ 0.0224 0.311 0.561 288.081 189.550 155.146 0.0222 0.304 0.572 200.318 166.202 181.491 0.0222 0.304 0.570 194.753 163.174 79.769 0.0222 0.304 0.573 196.179 171.051 252.632 0.0221 0.302 0.565 212.442 50.977 322.489 m1 /meV m2 /meV m3 /meV mββ /meV 11.642 14.482 51.705 4.422 38.357 39.312 63.153 14.161 7.772 11.602 50.757 3.291 28.017 29.312 57.456 9.503 21.954 23.583 54.707 19.573 q θ12 q θ13 q θ23 q δCP /◦ 0.227 0.00349 0.0424 69.137 0.227 0.00350 0.0402 69.230 0.227 0.00350 0.0401 69.489 0.227 0.00349 0.0398 69.273 0.227 0.00350 0.0397 68.679 mu /mc mc /mt md /ms ms /mb 0.00194 0.00279 0.0519 0.0183 0.00194 0.00282 0.0501 0.0181 0.00189 0.00283 0.0502 0.0181 0.00192 0.00283 0.0506 0.0182 0.00212 0.00283 0.0512 0.0179 mb /mτ 0.743 0.743 0.743 0.743 0.744 χ2l χ2q χ2total 12.213 12.474 24.688 0.0181 0.0286 0.0466 0.0353 0.0403 0.0756 0.00136 0.332 0.333 0.556 0.736 1.293 Table 6. The best fit values of the free parameters and the corresponding predictions for lepton and quark mixing parameters and fermion masses for the phenomenologically viable models of Type-I (continuation of table 5). – 51 – JHEP04(2021)239 Model Model I13 I14 I15 I16 I17 l sin2 θ13 2 l sin θ12 l sin2 θ23 l δCP /◦ α21 /◦ α31 /◦ 0.0224 0.310 0.564 298.913 348.080 154.909 0.0222 0.304 0.566 174.478 126.608 253.995 0.0221 0.304 0.575 198.634 33.969 191.453 0.0225 0.305 0.563 218.550 14.995 210.783 0.0223 0.302 0.575 228.392 28.967 162.487 m1 /meV m2 /meV m3 /meV mββ /meV 16.820 18.897 53.113 17.258 3.252 9.207 50.465 1.183 11.703 14.532 51.558 10.917 28.266 29.549 56.920 27.020 21.619 23.272 54.570 21.723 q θ12 q θ13 q θ23 q δCP /◦ 0.227 0.00350 0.0411 69.380 0.228 0.00343 0.0423 68.680 0.227 0.00350 0.0403 69.604 0.228 0.00350 0.0402 69.916 0.227 0.00346 0.0402 69.230 mu /mc mc /mt md /ms ms /mb 0.00195 0.00282 0.0518 0.0182 0.00208 0.00199 0.0592 0.0194 0.00192 0.00281 0.0544 0.0179 0.00232 0.00283 0.0569 0.0184 0.00258 0.00284 0.0577 0.0183 mb /mτ 0.743 0.743 0.744 0.744 0.743 χ2l χ2q 2 χtotal 14.858 2.081 16.939 1.091 64.251 65.342 0.0358 0.559 0.595 2.212 1.621 3.833 1.451 2.594 4.045 Table 7. The best fit values of the free parameters and the corresponding predictions for lepton and quark mixing parameters and fermion masses for the phenomenologically viable models of Type-I (continuation of table 6). – 52 – JHEP04(2021)239 Re(τ ) 0.4092 0.04232 −0.3716 0.1069 −0.03262 Im(τ ) 0.984 1.081 1.602 1.804 1.763 |αN1 /αN3 | 6.71 × 10−4 7.575 × 10−5 0.800 3.402 × 10−4 3.246 × 10−4 arg(αN1 /αN3 )/π 1.241 1.569 0.677 0.883 1.204 |αN2 /αN3 | 1.432 1.765 1.346 0.852 1.570 arg(αN2 /αN3 )/π 0.124 1.997 0.680 1.694 0.868 |αν2 /αν1 | 2.149 2.181 2.782 1.542 1.534 arg(αν2 /αν1 )/π 0.221 0.0199 1.821 0.282 0.633 αd1 /αd3 0.0339 0.0417 0.00262 0.00337 0.00285 αd2 /αd3 0.0519 0.00306 0.465 0.0299 0.00607 αd0 1 /αd3 −0.0302 −0.0181 4.809 × 10−4 0.00171 5.56 × 10−4 |αd0 2 /αd3 | 0.0173 0.00136 0.409 0.0263 0.0228 arg(αd0 2 /αd3 )/π 1.978 0.0906 0.997 0.732 0.927 |αd0 3 /αd3 | 0.541 0.440 0.267 0.566 0.106 arg(αd0 3 /αd3 )/π 0.420 0.456 1.949 1.589 0.755 |αu1 /αu3 | 0.0578 16.543 0.0605 0.0562 0.0636 arg(αu1 /αu3 )/π 0.703 0.385 0.640 0.230 1.658 |αu2 /αu3 | 0.843 4.735 × 10−4 1.075 1.369 0.924 arg(αu2 /αu3 )/π 1.034 1.306 0.778 0.232 1.753 |αu4 /αu3 | 233.679 112.554 365.736 94.717 83.623 arg(αu4 /αu3 )/π 1.678 1.465 1.302 0.731 1.644 (αν21 vu2 /(αN3 Λ))/meV 6.765 2.950 5.627 14.762 11.058 (αd3 vd )/GeV 0.490 0.661 0.746 0.752 1.033 (αu3 vu )/GeV 0.376 0.497 0.575 3.361 3.484 II 1 II 2 Re(τ ) Im(τ ) |αν2 /αν1 | arg(αν2 /αν1 )/π |αν3 /αν1 | arg(αν3 /αν1 )/π αd1 /αd3 αd2 /αd3 |αd4 /αd3 | arg(αd4 /αd3 )/π αd0 1 /αd3 |αd0 2 /αd3 | arg(αd0 2 /αd3 )/π |αd0 3 /αd3 | arg(αd0 3 /αd3 )/π |αd0 4 /αd3 | arg(αd0 4 /αd3 )/π |αu1 /αu3 | arg(αu1 /αu3 )/π |αu2 /αu3 | arg(αu2 /αu3 )/π (αν21 vu2 /(αN1 Λ))/meV (αd3 vd )/GeV (αu3 vu )/GeV −0.1342 3.095 0.326 0.368 1.685 1.992 17.909 1.740 1968.751 1.535 1.370 0.605 0.00905 0.977 1.014 1160.266 1.960 1.675 0.884 1.674 0.884 148.663 0.0194 17.414 −0.3947 3.092 0.378 4.329 × 10−4 0.0148 1.721 0.0538 0.101 100.642 0.291 −0.0472 0.0176 0.326 1.037 0.638 33.443 0.301 2.080 1.146 2.082 1.146 1.755 × 105 0.357 14.004 l sin2 θ13 2 l sin θ12 l sin2 θ23 l δCP /◦ α21 /◦ α31 /◦ 0.0224 0.310 0.553 4.115 333.672 162.827 0.0224 0.308 0.564 99.580 146.058 337.581 m1 /meV m2 /meV m3 /meV mββ /meV 74.100 74.599 89.918 69.038 12.512 15.190 51.850 5.050 q θ12 q θ13 q θ23 q δCP /◦ 0.227 0.00342 0.0436 67.809 0.227 0.00350 0.0380 68.868 mu /mc mc /mt md /ms ms /mb 0.00162 0.00278 0.0491 0.0192 0.00200 0.00284 0.0504 0.0181 mb /mτ 0.743 0.743 χ2l χ2q 2 χtotal 66.983 31.377 98.359 16.919 10.909 27.828 Table 8. The best fit values of the free parameters and the corresponding predictions for lepton and quark mixing parameters and fermion masses for the phenomenologically viable models of Type-II. – 53 – JHEP04(2021)239 Model Model V1 V2 V3 V4 V5 l sin2 θ13 2 l sin θ12 l sin2 θ23 l δCP /◦ α21 /◦ α31 /◦ 0.0221 0.300 0.556 197.938 166.227 283.237 0.0222 0.306 0.574 211.155 172.943 45.179 0.0223 0.303 0.542 250.695 299.647 136.764 0.0221 0.305 0.476 270.755 0.00786 180.423 0.0221 0.304 0.576 178.702 343.091 211.606 m1 /meV m2 /meV m3 /meV mββ /meV 12.953 15.555 51.759 3.987 2.333 8.924 50.202 1.05 × 10−4 18.512 20.418 53.496 17.629 104.520 104.874 115.832 104.875 115.424 115.745 125.851 109.795 q θ12 q θ13 q θ23 q δCP /◦ 0.227 0.00350 0.0416 78.674 0.227 0.00350 0.0416 78.216 0.227 0.00352 0.0392 61.068 0.228 0.00348 0.0411 66.222 0.227 0.00350 0.0403 69.271 mu /mc mc /mt md /ms ms /mb 0.0 0.00227 0.0675 0.0202 0.0 0.00224 0.0660 0.0200 0.00198 0.00299 0.0939 0.0191 0.00270 0.00245 0.00730 0.0173 0.00197 0.00282 0.0532 0.0174 mb /mτ 0.742 0.744 0.743 0.739 0.743 χ2l χ2q 2 χtotal 0.884 57.883 58.767 0.317 56.854 57.171 6.410 61.137 67.547 31.283 64.752 96.034 0.655 1.025 1.679 Table 9. The best fit values of the free parameters and the corresponding predictions for lepton and quark mixing parameters and fermion masses for the phenomenologically viable models of Type-V. – 54 – JHEP04(2021)239 Re(τ ) 0.4670 −0.4818 0.04772 −0.003098 −0.3313 Im(τ ) 1.250 1.232 1.021 1.007 1.618 |αN2 /αN1 | 171.272 5.059 1.917 × 107 7.571 × 105 1.181 × 107 arg(αN2 /αN1 )/π 0.203 0.723 0.157 1.146 1.987 |αN3 /αN1 | 8.270 94.999 9. × 105 5.692 × 105 4.117 × 106 arg(αN3 /αN1 )/π 1.857 1.081 1.156 1.120 1.924 αν2 /αν1 7.255 2.597 8.209 × 104 1042.230 5765.608 αν3 /αν1 367.080 26.949 1820.800 762.010 2131.981 αd1 /αd3 0.00213 0.00199 0.173 0.475 0.00201 αd2 /αd3 0.485 0.0221 0.00820 0.00572 0.0229 αd0 1 /αd3 4.719 × 10−4 4.351 × 10−4 −0.0782 −0.449 3.534 × 10−4 |αd0 2 /αd3 | 0.490 0.0415 0.00203 0.00574 0.0596 arg(αd0 2 /αd3 )/π 0.975 1.319 1.991 0.600 1.194 |αd0 3 /αd3 | 0.375 0.573 0.269 0.348 0.223 arg(αd0 3 /αd3 )/π 1.979 0.409 1.482 0.0353 0.563 |αu1 /αu3 | 0.0143 0.0139 3.424 44.172 0.0462 arg(αu1 /αu3 )/π 1.993 1.739 0.885 1.675 1.519 |αu2 /αu3 | 0.0609 0.0627 231.147 1.61 × 104 0.798 arg(αu2 /αu3 )/π 0.853 1.702 0.696 1.866 0.550 |αu4 /αu3 | — — 4342.700 3.252 × 105 360.682 arg(αu4 /αu3 )/π — — 1.608 0.0393 1.062 (αν21 vu2 /(αN1 Λ))/meV 6.693 11.659 14.799 104.887 115.765 (αd3 vd )/GeV 0.610 0.646 0.673 0.555 0.964 (αu3 vu )/GeV 73.344 71.808 0.0144 1.868 × 10−4 0.602 V6 V7 V8 V9 V10 Re(τ ) Im(τ ) |αN2 /αN1 | arg(αN2 /αN1 )/π |αN3 /αN1 | arg(αN3 /αN1 )/π αν2 /αν1 αν3 /αν1 αd1 /αd3 αd2 /αd3 αd0 1 /αd3 |αd0 2 /αd3 | arg(αd0 2 /αd3 )/π |αd0 3 /αd3 | arg(αd0 3 /αd3 )/π |αu1 /αu3 | arg(αu1 /αu3 )/π |αu2 /αu3 | arg(αu2 /αu3 )/π |αu4 /αu3 | arg(αu4 /αu3 )/π (αν21 vu2 /(αN1 Λ))/meV (αd3 vd )/GeV (αu3 vu )/GeV 0.09874 1.835 1619.521 1.994 8701.305 0.755 32.356 74.263 0.00400 0.0362 0.00209 0.0315 1.264 0.783 0.341 0.0488 0.746 1.639 0.746 94.958 0.128 25.174 0.625 3.580 −0.4687 1.850 2.779 × 104 1.908 2.797 × 104 1.711 374.097 105.143 0.00339 0.0345 0.00179 0.0250 0.766 0.588 1.596 0.0497 0.230 1.751 0.160 101.803 1.864 28.405 0.737 3.452 −0.02455 1.049 7.979 × 104 0.998 1.405 × 104 1.645 255.583 138.335 0.0549 0.00608 −0.0252 0.00240 0.127 0.703 0.367 16.826 0.668 4752.550 1.164 9.489 × 104 1.438 34.814 0.544 6.973 × 10−4 0.4915 1.598 4.37 × 107 1.031 9.499 × 106 0.0975 6894.423 2930.630 0.00204 0.406 0.00107 0.435 0.963 0.272 0.0577 0.0559 0.0788 0.970 1.645 369.490 0.131 60.161 0.740 0.566 −0.4562 1.790 2.863 × 104 1.002 1562.126 0.211 178.981 36.906 0.00390 0.0107 0.00198 0.0373 0.865 0.772 0.344 0.0607 1.700 1.248 1.305 94.852 0.841 50.645 0.630 3.259 l sin2 θ13 2 l sin θ12 l sin2 θ23 l δCP /◦ α21 /◦ α31 /◦ 0.0222 0.304 0.570 190.204 2.343 196.579 0.0222 0.304 0.574 187.373 359.144 200.631 0.0221 0.304 0.574 275.792 13.641 66.372 0.0222 0.304 0.565 167.688 15.279 222.357 0.0221 0.302 0.544 123.193 4.521 182.826 m1 /meV m2 /meV m3 /meV mββ /meV 24.370 25.847 55.752 23.026 27.659 28.969 57.302 26.167 27.984 29.280 57.446 26.792 53.675 54.361 73.398 51.553 43.265 44.115 66.400 43.174 q θ12 q θ13 q θ23 q δCP /◦ 0.227 0.00348 0.0402 69.170 0.227 0.00347 0.0407 67.896 0.228 0.00348 0.0410 67.305 0.227 0.00352 0.0406 69.753 0.227 0.00350 0.0374 68.068 mu /mc mc /mt md /ms ms /mb 0.00222 0.00283 0.0536 0.0182 0.00208 0.00282 0.0528 0.0181 0.00566 0.00255 0.0563 0.0186 0.00202 0.00278 0.0535 0.0183 0.00238 0.00282 0.0568 0.0184 mb /mτ 0.743 0.743 0.743 0.741 0.744 χ2l χ2q 2 χtotal 0.115 0.505 0.620 0.170 1.147 1.316 8.539 47.053 55.592 1.695 1.078 2.774 11.734 19.458 31.192 Table 10. The best fit values of the free parameters and the corresponding predictions for lepton and quark mixing parameters and fermion masses for the phenomenologically viable models of Type-V (continuation of table 9). – 55 – JHEP04(2021)239 Model V11 V12 V13 V14 Re(τ ) Im(τ ) |αN2 /αN1 | arg(αN2 /αN1 )/π |αN3 /αN1 | arg(αN3 /αN1 )/π αν2 /αν1 αν3 /αν1 αd1 /αd3 αd2 /αd3 αd0 1 /αd3 |αd0 2 /αd3 | arg(αd0 2 /αd3 )/π |αd0 3 /αd3 | arg(αd0 3 /αd3 )/π |αu1 /αu3 | arg(αu1 /αu3 )/π |αu2 /αu3 | arg(αu2 /αu3 )/π |αu4 /αu3 | arg(αu4 /αu3 )/π (αν21 vu2 /(αN1 Λ))/meV (αd3 vd )/GeV (αu3 vu )/GeV 0.4578 1.863 4.766 × 105 0.992 2.698 × 107 1.859 707.032 4982.583 0.00370 0.429 0.00192 0.452 1.003 0.327 1.967 0.0555 1.426 1.890 1.119 107.107 0.974 71.415 0.723 3.368 −0.04235 1.018 67.594 0.497 1.746 × 104 0.187 777.226 1.362 × 104 0.0354 0.00487 −0.0161 9.101 × 10−4 0.0239 0.0844 0.883 8.723 0.318 1435.860 0.583 2.785 × 104 0.580 0.00272 0.825 0.00223 0.4701 0.947 3040.620 1.868 5427.280 1.937 1801.840 1480.650 0.0123 0.0653 0.00270 0.0406 1.320 1.237 1.851 0.136 0.664 0.806 1.787 104.861 0.557 0.0281 0.268 1.191 −0.4983 0.881 855.157 1.025 2436.021 0.164 544.650 390.182 2.46 × 10−4 157.517 −0.419 12.948 1.027 2.414 1.097 0.0809 0.358 771.945 0.358 35.988 1.955 0.182 0.00474 1.318 l sin2 θ13 2 l sin θ12 l sin2 θ23 l δCP /◦ α21 /◦ α31 /◦ 0.0222 0.304 0.568 243.693 7.601 190.060 0.0222 0.304 0.573 194.576 323.991 33.404 0.0222 0.303 0.532 186.177 357.444 185.770 0.0218 0.303 0.573 274.541 7.250 187.830 m1 /meV m2 /meV m3 /meV mββ /meV 65.130 65.697 82.172 64.656 23.735 25.250 55.493 23.847 29.247 30.489 58.092 27.677 62.580 63.170 80.100 63.030 q θ12 q θ13 q θ23 q δCP /◦ 0.228 0.00346 0.0402 69.686 0.227 0.00350 0.0407 69.162 0.227 0.00329 0.0400 66.103 0.227 0.00346 0.0413 67.777 mu /mc mc /mt md /ms ms /mb 0.00251 0.00281 0.0533 0.0185 0.00194 0.00265 0.0507 0.0189 0.00361 0.00295 0.0488 0.0258 0.00289 0.00281 0.00847 0.0147 mb /mτ 0.742 0.743 0.763 0.737 χ2l χ2q 2 χtotal 3.092 1.431 4.523 0.0116 3.228 3.240 6.627 73.675 80.302 9.042 64.986 74.028 Table 11. The best fit values of the free parameters and the corresponding predictions for lepton and quark mixing parameters and fermion masses for the phenomenologically viable models of Type-V (continuation of table 10). – 56 – JHEP04(2021)239 Model V15 V16 V17 V18 Re(τ ) Im(τ ) |αN2 /αN1 | arg(αN2 /αN1 )/π αν2 /αν1 αν3 /αν1 αd1 /αd3 αd2 /αd3 |αd4 /αd3 | arg(αd4 /αd3 )/π αd0 1 /αd3 |αd0 2 /αd3 | arg(αd0 2 /αd3 )/π |αd0 3 /αd3 | arg(αd0 3 /αd3 )/π |αd0 4 /αd3 | arg(αd0 4 /αd3 )/π |αu1 /αu3 | arg(αu1 /αu3 )/π |αu2 /αu3 | arg(αu2 /αu3 )/π (αν21 vu2 /(αN1 Λ))/meV (αd3 vd )/GeV (αu3 vu )/GeV 0.03809 1.067 2.837 × 105 0.886 346.937 2274.630 0.0718 0.00697 0.412 0.612 −0.0342 0.00156 0.0384 0.167 1.362 0.617 0.487 2.093 × 10−4 1.442 0.0659 1.133 8.155 0.483 53.878 −0.02129 1.074 1.745 × 107 0.884 1.324 × 104 3644.926 0.0107 0.0316 1.964 1.265 0.00503 0.0985 0.883 1.080 1.714 0.171 0.346 2.113 × 10−4 1.647 0.0690 0.647 8.284 0.261 52.974 0.03559 1.041 3.18 × 106 1.090 1.129 × 104 180.796 0.00398 0.0285 0.598 1.153 0.00223 0.0323 0.605 2.105 2.579 × 10−4 0.231 1.436 2.506 × 10−4 0.417 0.0707 1.362 54.715 0.557 48.253 0.01418 1.056 3.461 × 107 1.010 5535.626 1.08 × 104 0.00370 0.0161 0.730 1.324 0.00199 0.0446 0.825 0.0843 0.774 0.105 0.142 2.162 × 10−4 1.481 0.0711 0.402 13.468 0.598 50.136 l sin2 θ13 2 l sin θ12 l sin2 θ23 l δCP /◦ α21 /◦ α31 /◦ 0.0222 0.299 0.570 269.281 207.895 236.478 0.0222 0.299 0.570 270.635 206.929 237.649 0.0222 0.301 0.571 224.199 322.312 149.590 0.0222 0.304 0.573 197.099 319.494 319.034 m1 /meV m2 /meV m3 /meV mββ /meV 1.591 8.760 50.349 0.636 1.574 8.757 50.350 0.630 1.482 8.740 50.178 3.633 16.768 18.851 52.898 16.671 q θ12 q θ13 q θ23 q δCP /◦ 0.227 0.00350 0.0403 68.824 0.227 0.00350 0.0403 68.798 0.227 0.00350 0.0404 71.046 0.227 0.00349 0.0402 69.213 mu /mc mc /mt md /ms ms /mb 0.00219 0.00275 0.0607 0.0185 0.00215 0.00275 0.0603 0.0185 0.00253 0.00275 0.0514 0.0183 0.00193 0.00282 0.0505 0.0182 mb /mτ 0.743 0.743 0.743 0.743 χ2l χ2q 2 χtotal 7.374 3.424 10.798 7.660 3.155 10.816 1.098 1.968 3.067 1.863 × 10−5 4.839 × 10−4 5.026 × 10−4 Table 12. The best fit values of the free parameters and the corresponding predictions for lepton and quark mixing parameters and fermion masses for the phenomenologically viable models of Type-V (continuation of table 11). – 57 – JHEP04(2021)239 Model Model V19 V20 V21 V22 V23 l sin2 θ13 2 l sin θ12 l sin2 θ23 l δCP /◦ α21 /◦ α31 /◦ 0.0222 0.301 0.572 223.846 321.694 151.525 0.0218 0.304 0.576 218.864 38.491 121.364 0.0219 0.307 0.571 233.313 1.592 148.807 0.0222 0.304 0.573 175.930 3.722 294.686 0.0222 0.303 0.566 239.595 6.776 190.708 m1 /meV m2 /meV m3 /meV mββ /meV 1.433 8.732 50.174 3.534 16.202 18.350 52.753 16.671 76.550 77.033 91.203 76.516 44.235 45.066 66.847 44.278 76.708 77.191 91.779 75.775 q θ12 q θ13 q θ23 q δCP /◦ 0.227 0.00350 0.0404 71.153 0.227 0.00348 0.0402 69.952 0.227 0.00347 0.0415 68.660 0.227 0.00346 0.0401 67.833 0.227 0.00354 0.0403 68.989 mu /mc mc /mt md /ms ms /mb 0.00254 0.00274 0.0511 0.0184 0.00243 0.00283 0.0640 0.0196 0.0 0.00275 0.0527 0.0184 0.0 0.00281 0.0488 0.0183 0.0 0.00282 0.0628 0.0184 mb /mτ 0.744 0.748 0.743 0.744 0.745 χ2l χ2q 2 χtotal 1.080 2.048 3.128 1.094 7.551 8.646 2.812 14.996 17.808 0.786 10.627 11.413 2.721 14.460 17.181 Table 13. The best fit values of the free parameters and the corresponding predictions for lepton and quark mixing parameters and fermion masses for the phenomenologically viable models of Type-V (continuation of table 12). – 58 – JHEP04(2021)239 Re(τ ) 0.01168 0.03374 −0.4722 −0.02250 0.4935 Im(τ ) 1.052 1.000 1.997 1.644 0.932 |αN2 /αN1 | 3.413 × 107 1.429 × 106 1.445 × 107 2.521 × 105 1.38 × 107 arg(αN2 /αN1 )/π 1.087 1.734 1.142 1.773 0.912 αν2 /αν1 7261.654 2467.673 3033.538 422.269 3529.148 αν3 /αν1 3004.292 1032.908 5167.630 719.326 4260.711 αd1 /αd3 5.973 × 10−6 1.462 × 10−5 0.00428 0.00272 4.274 × 10−6 αd2 /αd3 0.00236 0.00753 0.122 0.0821 2.754 × 10−6 |αd4 /αd3 | 0.317 0.687 3.278 1.146 0.252 arg(αd4 /αd3 )/π 0.472 1.160 0.811 0.445 0.759 αd0 1 /αd3 0.0166 0.0843 0.00224 0.00149 −0.00304 |αd0 2 /αd3 | 6.002 × 10−4 0.00165 0.122 0.0287 0.0108 arg(αd0 2 /αd3 )/π 1.823 3.134 × 10−6 0.921 0.750 1.945 |αd0 3 /αd3 | 0.792 0.360 0.252 0.481 0.267 arg(αd0 3 /αd3 )/π 0.546 1.658 1.341 0.302 0.0588 |αd0 4 /αd3 | 0.0390 0.455 1.400 1.840 0.0670 arg(αd0 4 /αd3 )/π 0.497 0.657 0.689 1.979 0.00297 |αu1 /αu3 | 2.391 × 10−4 2.973 × 10−4 2.922 × 10−5 0.00289 6.914 × 10−7 arg(αu1 /αu3 )/π 1.492 1.552 0.285 1.951 1.683 |αu2 /αu3 | 0.0681 0.0781 0.0525 0.0531 0.0134 arg(αu2 /αu3 )/π 0.773 0.807 0.285 0.275 1.684 (αν21 vu2 /(αN1 Λ))/meV 58.524 11.746 76.941 44.759 44.149 (αd3 vd )/GeV 1.249 0.478 0.810 0.545 1.345 (αu3 vu )/GeV 50.653 41.087 476.818 225.459 150.072 Model V24 V26 −0.03872 1.042 6260.908 0.166 5562.698 8929.744 0.00622 0.0396 0.686 1.657 0.00339 0.0724 1.263 0.770 0.319 0.592 1.266 2.24 × 10−4 1.945 0.0715 0.995 0.00254 0.350 48.477 V27 V28 0.03142 −0.05690 −0.4614 1.013 1.554 0.894 423.935 2.772 × 104 1.419 × 104 1.054 1.999 1.834 585.205 9996.838 8367.220 1441.682 4377.767 5285.115 6. × 10−5 6.512 × 10−4 1.667 × 10−6 0.00597 0.0298 8.085 × 10−4 0.852 1.055 0.0265 1.079 1.464 1.624 −0.0708 6.929 × 10−4 0.00229 0.00133 0.0632 0.00823 1.949 1.201 1.788 0.0522 0.721 0.219 0.284 0.402 1.999 0.158 1.405 0.106 0.172 1.488 1.674 2.838 × 10−4 0.00464 1.134 × 10−4 0.395 0.607 3.68 × 10−7 0.0757 0.0464 0.0102 1.718 1.583 1.546 0.0149 0.00556 0.00923 0.582 0.556 1.683 43.309 186.139 173.670 l sin2 θ13 2 l sin θ12 l sin2 θ23 l δCP /◦ α21 /◦ α31 /◦ 0.0211 0.310 0.570 115.138 356.402 191.174 0.0222 0.304 0.573 197.255 31.817 32.473 0.0219 0.305 0.574 228.295 56.081 134.582 0.0224 0.287 0.562 192.586 180.297 204.586 0.0223 0.303 0.546 281.439 7.680 152.994 m1 /meV m2 /meV m3 /meV mββ /meV 72.900 73.407 88.025 72.959 16.091 18.252 52.687 16.965 16.350 18.480 52.834 16.003 2.551 8.984 51.269 1.889 86.066 86.496 99.688 85.494 q θ12 q θ13 q θ23 q δCP /◦ 0.228 0.00350 0.0412 74.606 0.227 0.00349 0.0401 69.217 0.227 0.00352 0.0402 69.709 0.227 0.00360 0.0420 80.995 0.227 0.00347 0.0417 68.018 mu /mc mc /mt md /ms ms /mb 0.0 0.00315 0.0802 0.0183 0.00193 0.00282 0.0505 0.0182 0.00248 0.00282 0.0643 0.0193 0.0 0.00219 0.0213 0.0166 0.0 0.00282 0.0505 0.0186 mb /mτ 0.772 0.743 0.746 0.742 0.742 1.544 7.021 8.565 4.392 86.609 91.000 11.684 16.723 28.407 χ2l χ2q 2 χtotal 20.900 57.067 77.966 −5 9.63 × 10 8.846 × 10−5 1.848 × 10−4 Table 14. The best fit values of the free parameters and the corresponding predictions for lepton and quark mixing parameters and fermion masses for the phenomenologically viable models of Type-V (continuation of table 13). – 59 – JHEP04(2021)239 Re(τ ) −0.06476 Im(τ ) 2.009 |αN2 /αN1 | 2.321 × 106 arg(αN2 /αN1 )/π 1.825 αν2 /αν1 1345.488 αν3 /αν1 1882.619 αd1 /αd3 1.065 × 10−4 αd2 /αd3 0.242 |αd4 /αd3 | 13.771 arg(αd4 /αd3 )/π 1.482 αd0 1 /αd3 −0.0841 |αd0 2 /αd3 | 0.0785 arg(αd0 2 /αd3 )/π 0.00263 |αd0 3 /αd3 | 1.131 arg(αd0 3 /αd3 )/π 1.052 |αd0 4 /αd3 | 5.016 arg(αd0 4 /αd3 )/π 1.549 |αu1 /αu3 | 0.00259 arg(αu1 /αu3 )/π 1.507 |αu2 /αu3 | 0.00973 arg(αu2 /αu3 )/π 0.460 (αν21 vu2 /(αN1 Λ))/meV 73.353 (αd3 vd )/GeV 0.296 (αu3 vu )/GeV 487.744 V25 VII 1 VII 2 VII 3 Re(τ ) Im(τ ) |αν3 /αν2 | arg(αν3 /αν2 )/π αd1 /αd3 αd2 /αd3 |αd4 /αd3 | arg(αd4 /αd3 )/π αd0 1 /αd3 |αd0 2 /αd3 | arg(αd0 2 /αd3 )/π |αd0 3 /αd3 | arg(αd0 3 /αd3 )/π |αd0 4 /αd3 | arg(αd0 4 /αd3 )/π |αu1 /αu3 | arg(αu1 /αu3 )/π |αu2 /αu3 | arg(αu2 /αu3 )/π (αν1 αν2 vu2 /(αN1 Λ))/meV (αd3 vd )/GeV (αu3 vu )/GeV 0.3358 1.207 0.739 1.992 0.0544 0.00406 0.861 1.019 −0.0292 0.00238 0.205 0.517 0.417 0.466 1.450 0.0622 0.832 2.223 × 10−4 0.0184 16.054 0.670 75.570 −0.2769 1.203 0.580 1.062 0.00557 0.0476 1.328 1.198 0.00207 0.0279 1.222 0.561 0.431 0.175 1.014 2.64 × 10−5 1.729 0.0591 1.720 22.798 0.565 80.743 0.4734 1.288 2.329 0.889 0.00769 0.101 1.200 1.080 0.00286 0.0559 1.176 1.220 0.272 0.640 1.186 6.853 × 10−5 1.417 0.0658 1.417 11.786 0.433 75.765 l sin2 θ13 2 l sin θ12 l sin2 θ23 l δCP /◦ φ/◦ 0.0223 0.303 0.586 92.870 42.167 0.0222 0.304 0.569 209.195 31.273 0.0222 0.315 0.514 278.939 199.262 m1 /meV m2 /meV m3 /meV mββ /meV 0.0 8.614 50.020 1.983 0.0 8.614 50.148 2.800 0.0 8.614 49.898 3.602 q θ12 q θ13 q θ23 q δCP /◦ 0.227 0.00364 0.0379 69.225 0.227 0.00357 0.0394 69.398 0.227 0.00362 0.0406 68.704 mu /mc mc /mt md /ms ms /mb 0.0 0.00266 0.0505 0.0146 0.0 0.00289 0.0489 0.0165 0.0 0.00280 0.0504 0.0168 mb /mτ 0.746 0.744 0.745 χ2l χ2q 2 χtotal 19.526 38.795 58.321 0.257 15.228 15.486 18.998 13.937 32.935 Table 15. The best fit values of the free parameters and the corresponding predictions for lepton and quark mixing parameters and fermion masses for the phenomenologically viable models of Type-VII. – 60 – JHEP04(2021)239 Model VII 4 VII 5 VII 6 Re(τ ) Im(τ ) |αN2 /αN1 | arg(αN2 /αN1 )/π αν2 /αν1 αd1 /αd3 αd2 /αd3 |αd4 /αd3 | arg(αd4 /αd3 )/π αd0 1 /αd3 |αd0 2 /αd3 | arg(αd0 2 /αd3 )/π |αd0 3 /αd3 | arg(αd0 3 /αd3 )/π |αd0 4 /αd3 | arg(αd0 4 /αd3 )/π |αu1 /αu3 | arg(αu1 /αu3 )/π |αu2 /αu3 | arg(αu2 /αu3 )/π (αν21 vu2 /(αN1 Λ))/meV (αd3 vd )/GeV (αu3 vu )/GeV 0.04682 1.198 3.397 × 104 0.143 101.846 0.00964 0.0187 2.282 0.663 0.00384 0.0832 1.116 0.562 1.220 1.607 0.281 0.0115 1.317 0.0750 0.809 36.346 0.284 79.709 −0.1587 1.068 1441.443 0.164 19.052 0.00815 0.0538 1.806 1.414 0.00308 0.0554 1.290 0.638 1.195 0.211 1.203 × 10−6 0.0132 0.228 0.0842 1.981 32.470 0.320 57.900 −0.001939 1.123 4902.592 0.0237 103.807 0.00759 0.0444 1.837 0.728 0.00283 0.0482 0.678 0.603 1.083 0.474 0.378 0.0128 1.100 0.119 1.710 25.200 0.368 63.689 l sin2 θ13 2 l sin θ12 l sin2 θ23 l δCP /◦ φ/◦ 0.0224 0.353 0.498 273.592 356.632 0.0220 0.349 0.458 160.552 317.125 0.0224 0.315 0.588 316.174 173.353 m1 /meV m2 /meV m3 /meV mββ /meV 0.0 8.614 49.889 1.858 0.0 8.614 49.849 3.283 0.0 8.614 49.954 2.955 q θ12 q θ13 q θ23 q δCP /◦ 0.227 0.00349 0.0403 70.791 0.227 0.00350 0.0413 68.560 0.227 0.00350 0.0413 68.603 mu /mc mc /mt md /ms ms /mb 0.0 0.00270 0.0533 0.0185 0.0 0.00243 0.0551 0.0196 0.0 0.00244 0.0508 0.0194 mb /mτ 0.747 0.741 0.743 χ2l χ2q 2 χtotal 38.651 12.051 50.703 49.559 26.907 76.466 21.435 25.291 46.727 Table 16. 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RESEARCH ARTICLE Transferring Desirable Genes from Agropyron cristatum 7P Chromosome into Common Wheat Mingjie Lu☯, Yuqing Lu☯, Huanhuan Li, Cuili Pan, Yong Guo, Jinpeng Zhang, Xinming Yang, Xiuquan Li, Weihua Liu*, Lihui Li* National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China a11111 ☯ These authors contributed equally to this work. * liuweihua@caas.cn (WL); lilihui@caas.cn (LL) Abstract OPEN ACCESS Citation: Lu M, Lu Y, Li H, Pan C, Guo Y, Zhang J, et al. (2016) Transferring Desirable Genes from Agropyron cristatum 7P Chromosome into Common Wheat. PLoS ONE 11(7): e0159577. doi:10.1371/ journal.pone.0159577 Editor: Aimin Zhang, Institute of Genetics and Developmental Biology, CHINA Received: April 14, 2016 Accepted: July 4, 2016 Published: July 26, 2016 Copyright: © 2016 Lu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the National Key Technology Support Program of China (Grant No. 2013BAD01B02, to LL), the National Natural Science Foundation of China (Grant No. 31271714, to YL), and the CAAS Innovation Team Project (to LL). 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. Wheat-Agropyron cristatum 7P disomic addition line II-5-1, derived from the distant hybridization between A. cristatum (2n = 4x = 28, PPPP) and the common wheat cv. Fukuhokomugi (Fukuho), displays numerous desirable agronomic traits, including enhanced thousand-grain weight, smaller flag leaf, and enhanced tolerance to drought. In order to transfer these traits into common wheat, II-5-1 was induced by 60Co-γ ray, leading to the creation of 18 translocation lines and three deletion lines. Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) indicated that multiple wheat chromosomes were involved in the translocation events, including chromosome 2A, 3A, 5A, 7A, 3B, 5B, 7B, 3D and 7D. A. cristatum 7P chromosome was divided into 15 chromosomal bins with fifty-five sequence-tagged site (STS) markers specific to A. cristatum 7P chromosome. Seven and eight chromosomal bins were located on 7PS and 7PL, respectively. The above-mentioned translocation and deletion lines each contained different, yet overlapping 7P chromosomal fragments, covering the entire A. cristatum 7P chromosome. Three translocation lines (7PT-13, 7PT-14 and 7PT-17) and three deletion lines (del-1, del-2 and del-3), which contained the common chromosomal bins 7PS1-3, displayed higher thousand-grain weigh than Fukuho, suggesting that potential genes conferring high thousand-grain weigh might be located on these chromosomal bins. Therefore, wheat-A. cristatum 7P translocation lines with elite traits will be useful as novel germplasms for wheat genetic improvement. Introduction Bread wheat (Triticum aestivum L.), serving as a major source of calories, is one of the most important crops in the world. However, genetic improvement of wheat for high yield and quality to meet the demand from an ever-growing population has become challenging, as wheat breeding has been bottlenecked by a decreasing genetic diversity [1,2]. Therefore, researchers are persistently seeking for novel germplasms with broad genetic diversity for wheat genetic improvement. PLOS ONE | DOI:10.1371/journal.pone.0159577 July 26, 2016 1 / 15 Desirable Genes from Agropyron cristatum 7P Chromosome One effective approach to create novel germplasms is to transfer desirable genes conferring superior agronomic traits into common wheat from its wild relatives. This approach has been successfully employed in wheat improvement. For example, yield-related genes located on Th. bessarabicum chromosome arm 2JS were transferred into common wheat through wheat-Th. bessarabicum translocation line T2JS-2BS
2BL [3]. The genes conferring longer spikes and more kernels, as well as the seed storage protein genes conferring positive effect on bread making quality, were transferred into common wheat from Dasypyrum villosum, respectively [4–5]. Two new wheat-Thinopyrum ponticum translocation lines RWG33 and RWG34 were found to carry Sr43, which was resistant to the Ug99 race complex [6]. Stem rust resistance genes SrTA10187 and SrTA10171 were both derived from Aegilops tauschii [7]. Two T1BL
1RS translocation lines derived from the crosses between common wheat cultivar and rye, showed high stripe rust resistance and good quality [8]. Taken together, a large number of desirable genes have been successfully transferred into common wheat, resulting in a plenty of novel wheat germplasm. Thus far, several translocation lines have been widely used in wheat breading, and the two typical examples were wheat-rye 1BL
1RS and wheat-Haynaldia villosa 6VS
6AL translocation lines [9–11]. Agropyron cristatum (L.) Gaertn. (2n = 4x = 28, PPPP), one of the most important wild relatives of wheat, harbours multiple desirable agronomic traits, such as enhanced grain number per spike, multiple floret per spikelet, resistance to diseases, and tolerance to abiotic stresses [12–15]. In order to exploit these traits and transfer them into common wheat, distance hybridization between the common wheat cv. Fukuhokomugi (Fukuho) and A. cristatum was carried out in 1990s [16–19]. F1 hybrids were obtained successfully, and then a series of wheat-A. cristatum disomic addition lines were acquired [20–22]. These wheat-A. cristatum disomic addition lines were backcrossed with Fukuho or self-pollinated for several generations, until all of them displayed stable agronomic traits. Thus far, several wheat-A. cristatum disomic addition lines have been induced, producing various translocation, substitution, deletion and introgression lines [15,23–25]. For example, wheat-A. cristatum 6P addition line 4844–12 was used as the fundamental material to produce numerous wheat-A. cristatum 6P derivatives, some of which contain genes conferring enhanced grain number per spike [15,26], enhanced fertile tiller number per plant [23]. It’s also reported that wheat-A. cristatum disomic 2P addition line II-9-3 was induced, producing various wheat-A. cristatum 2P derivatives with high resistance to powdery mildew [27]. The wheat-A. cristatum disomic 7P addition line II-5 was found to display small flag leaf, enhanced thousand-grain weight, tolerance to drought. In order to transfer these elite traits into common wheat, II-5-1 was irradiated with 60Co-γ. Various wheat-A. cristatum 7P derivatives were acquired, Among them 18 translocation lines and three deletion lines were studied in this project. Cytological and molecular methods were applied to figure out the genetic constitutions of the 21 lines; The A. cristatum 7P chromosome was divided into different chromosomal bins, constructing the physical map of A. cristatum 7P chromosome. Besides, the agronomic traits, especially spike traits, were evaluated, and the chromosomal bins with thousand-grain weight-controlling genes were determined. Therefore, this study will not only provide novel germplasm for wheat breeding but also help to understand the characterization of A. cristatum 7P chromosome. Materials and Methods Plant materials The wheat-A. cristatum 7P disomic addition line II-5-1 (2n = 44) was derived from the wide hybridization between A. cristatum accession Z559 (2n = 4x = 28, PPPP) and Triticum aestivum cv. Fukuhokomugi (Fukuho) (2n = 6x = 42, AABBDD). PLOS ONE | DOI:10.1371/journal.pone.0159577 July 26, 2016 2 / 15 Desirable Genes from Agropyron cristatum 7P Chromosome Irradiation of wheat-A. cristatum 7P disomic addition line II-5-1 The II-5-1 plants were irradiated with 60Co-γ ray at a dosage of 20 Gray (Gy) and a dose rate of 0.5 Gy/min, when the plants were at the booting stage [28]. All the irradiated plants were artificially emasculated and pollinated with fresh pollens from the recipient parent Fukuho. F1 seeds were harvested, and then planted to produce the BC1F1 and BC2F1 populations. Morphological indexes of II-5-1 at the germination stage One hundred seeds from II-5-1 and Fukuho, respectively, were placed on filter paper with an osmotic potential of -0.5Mpa induced by the polyethylene glycol solution (PEG-6000) as described by Michael and Kaufman [29] and Gholamin et al. [30]. Mock treatment was conducted by using water. Each experiment was conducted three times with three replicates each time. All Petri dishes were placed in a growth chamber for seven days at a temperature of 22 ± 2°C and 50% relative humidity. On the seventh day, multiple morphological indices including germination rate, germination index, coleoptile length, radicle number were recorded from 20 seedlings in each replication. Some morphological indices were calculated using the following formulas: Germination rate = Total number of seeds germinated / Total number of seeds used x 100% Relative germination rate = Germination rate in PEG / Germination rate in water Germination index was defined as: Germination index = 1.0 n1+6/7 n2+5/7 n3+4/7 n4+3/7 n5+2/7 n6+1/7 n7, where n1-n7 were germination rates from the first day to the seventh day. GISH and FISH analysis Genomic in situ hybridization (GISH) was performed to detect A. cristatum 7P chromosomal segments of II-5-1 as well as its derivative lines. The root tips of all the materials were harvested when they grew to about 2 cm, and the chromosomes were prepared as described by Song et al. [28]. Genomic DNA of A. cristatum and Fukuho was isolated by CTAB method [31], and they were used as probe and block, respectively. A. cristatum genomic DNA was labelled with digoxigenin-11-dUTP. Meiosis of II-5-1 were studied as described by Jauhar and Peterson [32]. Briefly, young spikes of II-5-1 at the pollen mother cells (PMCs) metaphase I (MI) stage were fixed in Carnoy’s solution (6-ethanol: 3-chloroform: 1-acetic acid) for 24 h and stored at 4°C until use. GISH was performed on the root tip cells and PMCs as described by Liu et al. [33]. To identify the translocated wheat chromosomes, fluorescence in situ hybridization (FISH) was employed using repetitive DNA clones pAs1 and pHvG39 as probes, respectively [34]. A. cristatum genomic DNA and the clone pAs1 were labeled by the Digoxigenin-Nick Translation Mix, while the clone pHvG39 was labeled by Biotin-Nick Translation Mix. All the hybridization signals were observed using an OLYMPUS AX80 fluorescence microscope (Olympus Optical Co., Tokyo, Japan) and captured with a CCD camera (Diagnostic Instruments, Sterling Heights, MI, USA). Construction of the physical map of 7P chromosome with STS markers Sequence-Tagged Site (STS) markers were developed based on A. cristatum transcriptome sequences, which were deposited in the NCBI Transcriptome Shotgun Assembly (TSA) archive under the accession number GBAU00000000 [35]. These STS markers were used to characterize A. cristatum chromosome 7P, and then physically mapped on the different chromosomal bins. A. cristatum and Fukuho were used as the positive and negative control, respectively. The PCR programme was one cycle at 94°C for 5 min, followed by 36 cycles at 94°C for 1 min, at PLOS ONE | DOI:10.1371/journal.pone.0159577 July 26, 2016 3 / 15 Desirable Genes from Agropyron cristatum 7P Chromosome annealing temperature for 1 min, and at 72°C for 1 min, with a final extension at 72°C for 10 min. The amplified PCR products were separated by polyacrylamide gel electrophoresis (PAGE) with an acrylamide concentration of 8% and stained with silver. Evaluation of agronomic traits Plant materials were planted in 2.0 m rows, spaced 30 cm apart in the Beijing experiment station. The agronomic traits evaluated included the spike traits (grain number per spike, spikelet per spike, thousand-grain weight, grain length and grain width), the size of flag leaf, and also resistance to drought stress. The spike traits were measured as previously described [23]. Flag leaf length and width were measured at the milk stage. Drought tolerance evaluation was carried out as previously described [36]. Briefly, II-5-1 and Fukuho were treated with 20% Polyethylene glycol-6000 (PEG) after culturing for 7 days in water, and PEG-induced phenotypes were scored after 48 h. Results Characteristics of the wheat-A. cristatum 7P disomic addition line II-5-1 II-5-1 was derived from the wide hybridization between A. cristatum and Fukuho, followed by self-pollination for six generations. GISH result showed that there were 44 chromosomes, including 42 wheat chromosomes and two A. cristatum chromosomes (Fig 1A). Chromosomal pairing behavior in the pollen mother cells (PMCs) of II-5-1 was observed, and the chromosomal configuration was 2n = 22 II; Besides, neither trivalents nor quadrivalents were observed (Fig 1B), suggesting that II-5-1 was a stable disomic addition line. Fig 1. Identification of the wheat-A. cristatum disomic addition lineII-5-1. a, GISH pattern of II-5-1 showing 42 wheat chromosomes (shown in blue) and two A. cristatum chromosomes (shown in green and pointed by arrows); b, 21 bivalents are shown at the PMCs metaphase I (MI) stage of II-5-1; c-d, The morphologies of grain length (c) and width (d) of II-5-1 and Fukuho; e, The morphology of flag leaf of II-5-1 and Fukuho; f, The phenotype of seedling induced by PEG of II-5-1 and Fukuho. doi:10.1371/journal.pone.0159577.g001 PLOS ONE | DOI:10.1371/journal.pone.0159577 July 26, 2016 4 / 15 Desirable Genes from Agropyron cristatum 7P Chromosome Table 1. Agronomic traits of wheat-A. cristatum addition line II-5-1 and Fukuho. Materials Thousandgrain weight Grain length Grain width Grain numbers per spike Spikelet numbers per spike Flag leaf length Flag leaf width Plantheight 1.37 ±0.08b 105.0±2.3a 20.5±2.0a 11.1 ±1.2a 93.0±3.7b 21.3±3.6a 10.6 ±0.9a II-5-1 45.6±0.5a 7.84 ±0.05a 3.17 ±0.04a 30.6±2.3b 17.9±0.7a 17.3 ±1.9b Fukuho 38.4±2.3b 6.65 ±0.10b 3.21 ±0.08a 63.5±5.0a 18.5±1.0a 24.6±2.5a 1.78 ±0.18a Fertile tiller numbers per plant Spike length Significant differences in the mean are indicated at the P < 0.05 (lowercase letters), based on T tests. doi:10.1371/journal.pone.0159577.t001 II-5-1 displayed stable agronomic traits under three years’ field observation. As shown in Table 1, slight difference in plant height was observed between II-5-1 and Fukuho, but there was no significant difference in fertile tiller number per plant and spike length. II-5-1 showed enhanced thousand-grain weight and grain length compared with its recurrent parent Fukuho. The grain number per spike of II-5-1 was lower than that of Fukuho, and there was no significant difference in grain width and spikelet numbers per spike. The morphologies of grain length and grain width in II-5-1 and Fukuho were shown in Fig 1C and 1D. As shown in Fig 1E and Table 1, II-5-1 possessed narrower and shorter flag leaf compared with that of Fukuho. As shown in Table 2, relative germination rate, germination index, coleoptile length, radicle number of II-5-1 were higher than those of Fukuho, and relative germination index, relative coleoptile length, relative radicle number of II-5-1 were also increased compared with Fukuho, respectively. At the seedling stage, II-5-1 displayed less severe wilting than Fukuho (Fig 1F). These results suggested that II-5-1 displayed higher tolerance to drought compared with Fukuho. In sum, we propose that multiple elite agronomic traits in II-5-1, such as enhanced thousand-grain weight, longer grain length and higher tolerance to drought might be attributed to desirable alien genes from the A. cristatum 7P chromosome. Cytological identification of wheat-A. cristatum 7P translocation and deletion lines In order to integrate the elite traits of II-5-1 into common wheat, wheat-A. cristatum 7P addition lineII-5-1 was irradiated with 60Co-γ ray, and then backcrossed with the recipient parent Fukuho. Then BC1F1 seeds harvested from the irradiated plants were backcrossed with Fukuho to obtain BC2F1 plants. 219 BC1F1 lines were acquired totally, among which 18 translocation lines and three deletion lines were further investigated by GISH and FISH. As shown in Table 3 and Fig 2, 18 translocation lines contained 42 chromosomes consisting of 41 wheat chromosomes, while three deletion lines contained 43 chromosomes consisting of 42 wheat chromosomes. 18 translocation lines were categorized into four translocation types, according to the relative size of the translocated chromosomal segments compared to either 7P chromosome arm (Fig 2 and Table 3). These four types were as follows: whole-arm translocation, large segmental translocation, small segmental translocation and intercalary translocation. Table 2. Morphological indices of II-5-1 and Fukuho induced by PEG at the germination stage. Relative Germination Rate Germination Index Relative Germination Index Coleoptile Length Relative Coleoptile Length Radicle Number Relative Radicle Number II-5-1 0.94 1.89±0.01a 0.85 23.4±1.1a 0.84 4.1±0.7a 0.95 Fukuho 0.88 1.83±0.01b 0.75 19.5±1.9b 0.71 3.3±0.5b 0.60 Significant differences in the mean are indicated at the P < 0.05 (lowercase letters), based on T tests. doi:10.1371/journal.pone.0159577.t002 PLOS ONE | DOI:10.1371/journal.pone.0159577 July 26, 2016 5 / 15 Desirable Genes from Agropyron cristatum 7P Chromosome Table 3. 18 wheat-A. cristatum 7P translocation lines and three deletion lines were acquired. Materials Chromosome number Translocated wheat chromosomes Translocation types 7P chromosome bins Generations 7PT-1 42 3D 7PS
3DL S1-7 BC1F1 7PT-2 42 2A 7PS-2AS
2AL S4-7 BC1F1 7PT-3 42 3B 7PS-3BS
3BL S5-7 BC1F1 7PT-4 42 5B 5BL
7PL L1-8 BC1F1 7PT-5 42 5A 5AS
5AL-7PL L4-8 BC1F1 7PT-6 42 3A 3AS
3AL-7PL L4-8 BC1F1 7PT-7 42 7A 7AS
7AL-7PL L5-8 BC1F1 7PT-8 42 7B 7BS
7BL-7PL L6-8 BC1F1 7PT-9 42 5A 5AS
5AL-7PL L8 BC1F1 7PT-10 42 7D 7DS
7DL-7PL-7DL L4-6 BC1F1 7PT-11 42 7A 7AS-7PS-7AS S2-4 BC1F1 7PT-12 42 5A 7PS
7PL-5AL S1-7, L1-7 BC1F1 7PT-13 42 7B 7PS
7PL-7BL S1-7, L1-5 BC1F1 7PT-14 42 7A 7PS
7PL-7AL S1-7, L1-4 BC1F1 7PT-15 42 3A 7PS
7PL-3AL S1-7, L1 BC1F1 7PT-16 42 3A 3AS-7PS
7PL S1-6, L1-8 BC1F1 7PT-17 42 5A 5AS-7PS
7PL S1-3, L1-8 BC1F1 7PT-18 42 3B 3BS-7PS
7PL S1-2, L1-8 BC1F1 del-1 43 N/A N/A S1-5, L1-8 BC1F1 del-2 43 N/A N/A S1-7, L1-2 BC1F1 del-3 43 N/A N/A S1-7 BC1F1 N/A: Not applicable doi:10.1371/journal.pone.0159577.t003 7PT-1 and 7PT-4 were both whole-arm translocation lines, in which translocation occurred to wheat chromosomes 3D and 5B, respectively. Seven small segmental translocation lines were identified (7PT-2, 7PT-6, 7PT-5, 7PT-9, 7PT-7, 7PT-3 and 7PT-8), in which translocation occurred on wheat chromosomes 2A, 3A, 5A, 5A, 7A, 3B and 7B, respectively. There were seven large segmental translocation lines (7PT-15, 7PT-12, 7PT-17, 7PT-14, 7PT-18, 7PT-16 and 7PT-13), which were translocated to 3A, 5A, 5A, 7A, 3B, 3A and 7B, respectively; 7PT-10 and 7PT-11 belonged to intercalary translocation lines, which were translocated to wheat chromosome 7DL and 7AS, respectively; However, only the short arm was left in 7PT-11, and the long arm was missing. GISH and FISH patterns of three translocation lines (7PT-8, 7PT-10 and 7PT-14) were shown in Fig 3, and GISH and FISH patterns of other translocation lines were shown in S1 Fig. Beside 18 translocation lines, there were three deletion lines (del-1, del-2 and del-3) identified. There was only one chromosome arm left in del-3, and the other chromosome arm was missing. There were partial chromosomal segments less than one chromosome arm missing in del-1 and del-2. Construction of the physical map of 7P chromosome We further constructed the physical map of A. cristatum 7P chromosome with STS markers anchored to each bin. A large number of STS markers were screened to find those that can successfully be amplified in both A. cristatum and II-5-1, and in at least one of the 21 translocation and deletion lines, but not in Fukuho, resulting in the discovery of 126 7P-specific STS markers. Among them, 55 STS markers displayed high amplification efficiency and reproducibility, and were chosen for downstream analysis (S1 Table). According to the co-occurrences of the STS PLOS ONE | DOI:10.1371/journal.pone.0159577 July 26, 2016 6 / 15 Desirable Genes from Agropyron cristatum 7P Chromosome Fig 2. Eighteen wheat-A.cristatum translocation lines and three deletion lines were identified. The left map showing the schematic diagram of A. cristatum chromosome 7P. Pink and blue color represented A. cristatum 7P and wheat chromosomal segments, respectively. Dotted line boxes indicated the deleted chromosomal segments of A. cristatum 7P chromosome. doi:10.1371/journal.pone.0159577.g002 markers and 7P chromosomal bins in these translocation and deletion lines, the 55 STS markers were mapped to different bins of A. cristatum chromosome 7P, with 30 and 25 STS markers located on 7PS and 7PL, respectively (Fig 4). The chromosomal bins of 7PS and 7PL near the centromere were named as 7PS1 and 7PL1, respectively; while the chromosomal bins near the ends were named as 7PS7 and 7PL8, respectively (Fig 4). Each chromosomal bin contained different STS markers, ranging from 1 to 12. The primer sequences of 55 STS markers were listed in S2 Table, and PCR amplification patterns of five markers were shown as examples in Fig 5. As shown in Fig 2 and Table 3, 18 translocation lines contained different chromosomal bins of 7PS and 7PL. Four translocation lines (7PT-1, 7PT-2, 7PT-3 and 7PT-11) only contained both 7PS or part of 7PS, and seven translocation lines (7PT-4, 7PT-5, 7PT-6, 7PT-7, 7PT-8, 7PT-9 and 7PT-10) contained both 7PL or part of 7PL. Four translocation lines (7PT-12, 7PT13, 7PT-14 and 7PT-15) contained 7PS and part of 7PL, while three translocation lines (7PT16, 7PT-17 and 7PT-18) contained 7PL and part of 7PS. The deletion line del-1 included the entire 7PL arm and 7PS1-5, del-2 included the entire 7PS and 7PL1-2, and del-3 only included the entire 7PS. From these results, we could infer that all the 7P chromosomal bins have been translocated onto wheat chromosomes. All these results indicated that various A. cristatum 7P PLOS ONE | DOI:10.1371/journal.pone.0159577 July 26, 2016 7 / 15 Desirable Genes from Agropyron cristatum 7P Chromosome Fig 3. Three wheat-A. cristatum translocation lines identified by GISH and FISH. 7PT-8 was the small chromosomal segment translocation line, which was translocated to wheat chromosome 7B (Fig 3A and 3D). 7PT-10 was the small chromosomal segment translocation line, which was intercalated into wheat chromosome 7D (Fig 3B and 3E). 7PT-14 was the large chromosomal segment translocation line, which was translocated to wheat chromosome 7A (Fig 3C and 3F). FISH patterns were shown in a-c, in which the clone pHvG39 and pAs1 was labeled green and red, respectively. GISH patterns were shown in d-f, in which A. cristatum and wheat DNA was stained red and blue, respectively. doi:10.1371/journal.pone.0159577.g003 chromosomal bins were translocated onto 18 translocation lines and three deletion lines, and all these chromosomal bins can cover the entire A. cristatum 7P chromosome. Evaluation of spike traits of wheat-A. cristatum 7P translocation and deletion lines Agronomic traits, especially the spike traits, were investigated in six translocation (7PT-7, 7PT-8, 7PT-10, 7PT-13, 7PT-14 and 7PT-17) and three deletion lines (del-1, del-2 and del-3) at the BC2F1 generations. As shown in Table 4, three translcoation lines (7PT-13, 7PT-14 and 7PT-17) as well as three deletion lines displayed higher thousand-grain weight and longer grain length than those of Fukuho, respectively. However, there were no significant difference in both thousand-grain weight and grain length among other three translocation lines (7PT-7, 7PT-8 and 7PT-10) and Fukuho. There was no significant difference in grain width in all the nine lines as well as two parents. Besides, all the nine lines except 7PT-7 showed less grain number per spike than that of Fukuho, but some of them showed higher grain number per spike than that of II-5-1. There was no significant difference in spikelet number per spike PLOS ONE | DOI:10.1371/journal.pone.0159577 July 26, 2016 8 / 15 Desirable Genes from Agropyron cristatum 7P Chromosome Fig 4. The physical map of A. cristatum 7P chromosome with 55 STS markers. Left panel: 15 chromosomal bins of A. cristatum 7P chromosome; Right panel: Physical locations of STS markers specific to 7P chromosome. The words "Agc" in front of the STS markers are omitted. doi:10.1371/journal.pone.0159577.g004 PLOS ONE | DOI:10.1371/journal.pone.0159577 July 26, 2016 9 / 15 Desirable Genes from Agropyron cristatum 7P Chromosome Fig 5. PCR amplification patterns of five STS markers. a, Agc23527; b, Agc389; c, Agc52001; d, Agc27894; e, Agc51785. 1, Marker; 2, Z559; 3, II-5-1; 4, Fukuho; 5, 7PT-3; 6, 7PT-2; 7, 7PT-1; 8, del-3; 9, 7PT-15; 10, del-2; 11, 7PT-14; 12, 7PT-13; 13, 7PT-12; 14, 7PT-16; 15, del-1; 16, 7PT-17; 17, 7PT-18; 18, 7PT-4; 19, 7PT-5; 20, 7PT-6; 21, 7PT-7; 22, 7PT-8; 23, 7PT-9; 24, 7PT-10; 25, 7PT-11. doi:10.1371/journal.pone.0159577.g005 between two parents (II-5-1 and Fukuho), but all the nine lines except 7PT-7 and 7PT-10 contained more spikelet number per spike than that of either parent. As shown in Fig 6, the chromosomal bins 7PS1-3 were present in three translocation lines (7PT-13, 7PT-14 and 7PT-17) and three deletion lines but absent in other three translocation lines. All the results suggested that there might be genes conferring high thousand-grain weight and grain length on the chromosome bins 7PS1-3 of A. cristatum chromosome. Discussion Various translocation lines were produced by ionizing radiation There are large quantities of elite genes in the wild relatives of wheat that can be used to improve the yield, quality, disease resistance and stress tolerance in wheat breeding. Generally speaking, creation of wheat alien addition lines is the first key step to transfer these desirable genes into common wheat. However, wheat alien addition line could not be used directly due to its large alien chromosomal segments. Therefore, wheat alien addition lines are often used as the fundamental materials to produce translocation lines with small alien chromosomal segments. Table 4. Evaluation of the spike traits of some wheat-A. cristatum translocation and deletion lines. Materials Translocation types Thousand-grain weight Grain length Grain width Grain number per spike Spikelet numberper spike II-5-1 N/A 45.6±0.5a 7.84±0.05a 3.17±0.04a 30.6±2.3d 17.9±0.7d Fukuho N/A 38.4±2.3b 6.65±0.10d 3.21±0.08a 63.5±5.0a 18.5±1.0d 7PT-7 7AS
7AL-7PL 37.5±1.5b 6.74±0.08d 3.17±0.13a 60.5±7.8a 19.5±2.1bcd 7PT-8 7BS
7BL-7PL 36.9±4.6b 6.69±0.25d 3.28±0.08a 35.3±4.1d 20.8±1.3abc 7PT-10 7DS
7DL-7PL-7DL 37.6±3.4b 6.75±0.08d 3.34±0.08a 37.7±7.5cd 19.0±2.6cd 7PT-13 7PS
7PL-7BL 46.0±2.1a 7.38±0.08bc 3.27±0.17a 29.7±4.5d 20.7±0.6abc 7PT-14 7PS
7PL-7AL 43.7±1.5a 7.23±0.03bc 3.36±0.21a 50.5±7.8b 21.5±1.4ab 7PT-17 5AS-7PS
7PL 45.1±5.4a 7.33±0.04bc 3.35±0.20a 38.0±6.8cd 21.0±1.4abc del-1 N/A 45.7±4.5a 7.21±0.11c 3.30±0.21a 46.3±9.3bc 20.9±1.0abc del-2 N/A 44.0±2.7a 7.41±0.14b 3.27±0.19a 37.0±7.5cd 21.7±2.1ab del-3 N/A 46.6±1.7a 7.26±0.20bc 3.42±0.19a 48.5±4.9b 22.0±1.4a Significant differences in the mean are indicated at the P < 0.05 (lowercase letters), based on Duncan’s multiple range tests. doi:10.1371/journal.pone.0159577.t004 PLOS ONE | DOI:10.1371/journal.pone.0159577 July 26, 2016 10 / 15 Desirable Genes from Agropyron cristatum 7P Chromosome Fig 6. Six lines sharing one common chromosomal bins 7PS1-3 displayed high thousand-grain weight. doi:10.1371/journal.pone.0159577.g006 There were several methods to induce addition lines, such as gamma irradiation, CS ph1b system and gametocidal chromosome. Considering the low level of pairing between A. cristatum P genome and wheat ABD genomes, development of translocation lines by ionizing radiation seems to be a more appropriate approach [37]. Compared with other approaches, there’re much more chromosomal breakages by using this method, resulting in various translocation types [2]. In this study, 18 different wheat-A. cristatum 7P translocation lines and three deletion lines were obtained, which was mainly attributed to multiple chromosomal breakages caused by 60Co-γ irradiation. Besides, two intercalary translocation lines (7PT-10 and 7PT-11) were caused by double-strand chromosomal breakages. Therefore, ionizing radiation proved to be an effective way to produce translocation lines in this study. The effectiveness of gamma irradiation was also illustrated by several other examples. Various 6P and 2P translocation lines were produced from the wheat-A. cristatum 6P addition line 4844–12 [28] and 2P addition line II-9-3 by 60Co-γ irradiation [27], respectively. A series of structural aberrations involving Thinopyrum bessarabicum chromosome 4J were obtained through gamma radiation [38]. Plenty of wheat-Haynaldia villosa translocation lines with Pm21 were acquired by ionizing radiation [39]. Establishment of the physical map of 7P chromosome with STS markers Cytological methods such as GISH/FISH are often used to identify alien chromosomal segments. However, these methods are ineffective in identifying small chromosomal segments. Moreover, it is not feasible to screen a large number of translocation lines by GISH/FISH, due to the fact that chromosome preparation and hybridization is quite labor-intensive and timeconsuming. Compared with the cytological method, molecular marker is more effective to detect alien chromosomal segments. After genotyping the translocation line with molecular markers, we will get a general idea of the translocated chromosomal segments quickly. PLOS ONE | DOI:10.1371/journal.pone.0159577 July 26, 2016 11 / 15 Desirable Genes from Agropyron cristatum 7P Chromosome In this study, 18 different translocation lines and three deletion lines were characterized by GISH/FISH and 55 STS markers. A. cristatum 7P chromosome were divided into 15 chromosomal bins depending on the presence or absence of STS markers, leading to the construction of the physical map of 7P chromosome. Once the physical map of 7P chromosome was constructed, genes from this chromosome could be easily located on specific chromosomal bins. In this study, genes conferring enhanecd thousand-grain weight and grain length were preliminarily mapped on the chromosomal bins 7PS1-3. There’re other examples reported previously, showing the constructed physical maps with novel alien genes. For example, the physical map of Thinopyrum bessarabicum chromosome 4J consisted of 24 segmental blocks, and the bluegrained gene BaThb was mapped on the block 4JL-11 [38]. The physical map of A. cristatum 6P was built with 255 STS markers, and the leaf rust resistance genes were mapped on the region 6PS-0.81–1.00 [24]. Wheat-A. cristatum 7P translocation lines are potentially valuable in wheat breeding Grain number per spike, fertile tiller number per plant and thousand-grain weight are three main factors determining wheat yield. Among these three factors, thousand-grain weight is least affected by environment [40]. Therefore, it’s important to increase thousand-grain weight in order to stably enhance the wheat yield. Thousand-grain weight is mainly determined by grain width, length and thickness [41–44]. In this study, wheat-A. cristatum 7P addition line II-5-1 displayed enhanced thousand-grain weight and drought tolerance, and also a small flag leaf. It has been reported that a small flag leaf lead to higher photosynthetic rate of the whole plant and in turn higher grain weight [45,46]. A small flag leaf was also reported to reduce transpiration and enhance drought tolerance [47]. Whether the small size of the flag leaf in II-5-1 contributed to its high grain weight and drought tolerance remains to be investigated. Among the 18 translocation lines reported in this study, six translocation lines (7PT-7, 7PT-8, 7PT-10, 7PT-13, 7PT-14 and 7PT-17) and three deletion lines (del-1, del-2 and del-3) displayed good agronomic performance (especially seeds-setting). According to FISH results, the translocations in all six translocation lines except 7PT-17 occurred on the wheat chromosomes which belonged to wheat homoeologous group 7. These results suggested that wheat chromosome 5A, as well as 7B and 7A, conferred good homoeologous compensation with A. cristatum chromosome 7P. The good homoeologous compensation between 7P and 5A might be attributed by the genetic rearrangement of A. cristatum chromosome 7P, and the similar phenomenon was also observed on A. cristatum chromosome 6P [48]. Three translocation lines and three deletion lines with chromosomal bins 7PS1-3 showed increased thousand-grain weight compared with Fukuho, while three translocation lines without chromosomal bins 7PS1-3 displayed decreased thousand-grain weight. The results suggested that there may be some genes conferring high thousand-grain weight on 7PS1-3. However, the agronomic traits of other translocation lines couldn’t be evaluated due to their bad seeds-setting, which might be caused by irradiation or poor compensation. The specific chromosomal locations and identities of superior genes on the chromosomal bins 7PS1-3 conferring high thousand-grain weight still need further investigation. Therefore, to recover the genetic compensation and effectively utilize these lines in future breeding programs, we need to further reduce the linkage drag, preferably by back-crossing or ph1b system. Supporting Information S1 Fig. Fifteen wheat-A. cristatum translocation lines were identified by GISH and FISH. FISH patterns were shown in a1-o1, while GISH patterns were shown in a2-o2. a, 7PT-1; b, PLOS ONE | DOI:10.1371/journal.pone.0159577 July 26, 2016 12 / 15 Desirable Genes from Agropyron cristatum 7P Chromosome 7PT-2; c, 7PT-3; d, 7PT-4; e, 7PT-5; f, 7PT-6; g, 7PT-7; h, 7PT-9; i, 7PT-11; j, 7PT-12; k, 7PT13; l, 7PT-15; m, 7PT-16; n, 7PT-17; o, 7PT-18. (PDF) S1 Table. The PCR amplification results of 55 STS markers in all the materials. (XLS) S2 Table. The primers sequences of 55 STS markers. (XLS) Acknowledgments The authors would like to thank the cobalt source chamber of Beijing University for irradiation treatment. 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УДК [[783:271.2]:[784.1.087.684:78.082.1:78.071.1Луньов]](477)“20”(045) DOI: 10.18523/2617-8907.2023.6.65-71 Олена Яремчук ПОЛІСТИЛІСТИКА ПАРАЛІТУРГІЙНОЇ МУЗИКИ СВЯТОСЛАВА ЛУНЬОВА У статті простежено шлях розвитку православної культури в Україні, зокрема паралітургійної музики. Описано пошуки духовного в музиці, висвітлено ключові надихаючі обставини. Розглянуто розмаїття напрямів, стилів, тематики в хоровій симфонії, що репрезентує культурну панораму різних історичних періодів на території України. Досліджено паралітургійний напрям творчості сучасного українського композитора Святослава Луньова. Зокрема, проаналізовано спорідненість архаїчних культових піснеспівів із новітніми паралітургійними композиціями у творчості С. Луньова на прикладі симфонії для мішаного хору a cappella «Страсна седмиця». Ключові слова: полістилістика, паралітургійна музика, духовний, Страсна седмиця, молитва, обіход, композиторська творчість, Святослав Луньов. Постановка проблеми. Паралітургійна музика – надзвичайно потужний чинник формування української музичної культури, обумовлений зростанням і розвитком духовності соціуму. Часто паралітургійні твори композиторів входять до репертуарів різноманітних фестивалів, концертів, конкурсів, навчальних програм з диригування та хорового співу в закладах освіти музичного спрямування. Паралітургійна музика стала частиною творчості як «класиків української музики» – О. Кошиця, М. Леонтовича, К. Стеценка, Я. Яциневича та ін., так і сучасних композиторів – Л. Дичко, Г. Гаврилець, С. Луньова, В. Польової, В. Сильвестрова, В. Степурка та ін. Музика, зокрема паралітургійна, – одна з основних художніх проєкцій самосвідомості культури, адже вона є похідною від духовної традиції, яка є основою історико-­культурного коду нації. Яскравим прикладом звернення до паралітургійної музики серед сучасних українських композиторів є творчість С. Луньова, дослідженню особливостей якої присвячено цю статтю. Стан наукової розробки проблеми. Різно­ аспектному аналізу літургійних, позалітургійних та паралітургійних жанрів у богослужбовій музиці присвячено численні наукові розвідки, зокрема фундаментальні дослідження М. Антоненко (2020), Н. Александрової (2008), Н. Герасимової-Персидської (1999); праці Т. Гусарчук (2018), Г. Васильченко-Міхно (1993), А. Єфіменко (2010, 343–57), О. Зосім (2011, 297–304) та ін. Особливості паралітургійної музики В. Польової розглянуто в підрозділі дисертаційного дослідження А. Бакумець (2021, 175–95). Духовну творчість українських композиторів вивчає © Олена Яремчук, 2023 О. Козаренко (2002, 150–60). Особливостям музичної мови С. Луньова присвячено розділ дисертаційного дослідження О. Моцар (2016, 138–86). У статті Ж. Зубко (2011) розглянуто використання композитором засобів духовної музики, а саме псалмодійного речитативу та мелодекламації богослужбового тексту у творі «Дніпро» для інструментального складу. Наукові розвідки Ю. Воловчук (2018, 49–55) та Т. Кушніра (2020, 165–9) присвячено аналізу симфонії для мішаного хору a cappella «Страсна седмиця». Однак залишається нерозкритим питання місця цього твору та його стилістичних особливостей як одного зі зразків паралітургійної музики. Адже, як стверджує композитор і музикознавець Олександр Козаренко: «Так необхідне зараз “вгризання в семантику” музичної матерії для розуміння процесів, що її наповнюють, є, очевидно, лишень першою сходинкою до розкриття інтегрального змісту твору мистецтва (виконаного чи написаного), а відтак – розкриття певної його ролі у безкінечному процесі “духовного пригодництва”» (Козаренко 2012, 5). Мета статті зумовлена науковою новизною дослідження, оскільки стилістичні особливості симфонії для мішаного хору a cappella «Страсна седмиця» С. Луньова вперше стали предметом музично-культурологічного аналізу в контексті української паралітургійної музики. Важливо простежити зв’язок цього твору з архаїчними культовими піснеспівами на тлі історичного розвитку духовної музики. 66 ISSN 2617-8907. Наукові записки НаУКМА. Історія і теорія культури. 2023. Том 6 Виклад основного матеріалу. Відомо, що церковну музику складають канонічні піснеспіви, що виконуються під час богослужінь, – вечірні, літургії, деякі святкові служби, акафісти, панахиди, вінчання тощо. Вони супроводжуються як співом/мелодекламацією/псалмодіюванням священнослужителів, так і хором. Послідовність і тексти більшості з них в обіході з початку VI ст. Британський історик Норман Дейвіс у книжці «Європа. Історія» пише: «Зважаючи на те, що в Европі так і не було загальної для всіх усної мови, тобто, спільної словесної mousike, музичну мову Европи – її несловесне mousike – слід вважати за найтривалішу лінію її спільної культури. Справді, оскільки вона простягається від Іспанії до Росії, але не поширюється на Індію чи ісламський світ, виникає спокуса припустити, що музика – єдиний універсальний паневропейський засіб спілкування» (Дейвіс 2008, 136). Середземноморський культурний простір, зважаючи на його територіальне та культурно-культове минуле, своїм корінням сягає доби Стародавнього Риму. У результаті геополітичних змін наступницею східної частини Римської імперії стала Візантія. Саме у візантійському обряді формується система «осьмогласся» (подібна до середньовічних ладів григоріанського співу), в структурі якої існує вісім ладів літургійної музики, свій порядок використання в богослужбовій практиці послідовною зміною щотижня одного гласу на інший. Ця послідовність утворює восьмитижневе гласове коло, яке має назву «гласовий стовп». У VIII ст. її упорядкував в «Октоіх» Іоанн Дамаскін і згодом доопрацював Феодор Студит у IX ст. Канонічне музичне мистецтво на території сучасної України сягає корінням часів Київської Русі, а точніше – відлік починається з моменту її хрещення у 987–989 рр. Традиції служіння православної церкви були сформовані навколо візантійської, або східної, культури. Як зазначає музикознавець Юрій Ясіновський: «Разом із християнством Київська Русь перейняла з Візантії найрозвиненіший на той час літургійний спів, його форми, жанри, богословсько-етичну сутність, а також багату виконавську практику, систему вишколу професійних кадрів, писемні форми запису, музично-теоретичні основи. Візантійсько-християнська спадщина заклала міцні підвалини руської (української) писемної професійної музики» (Ясіновський 2015, 32). Прототипом невменної нотації, яка була основною системою запису церковних піснеспівів у середньовічній Європі, зокрема пройшла три етапи еволюції у Візантії, в Україні у ХІІ ст. є знаменний розспів, що побутував до XVI ст., зазнаючи трансформацій у нотуванні: від широкого вжитку людьми до використання священнослужителями та вузькопрофільними професійними співаками. Процес розвитку українського церковного співу впродовж тисячоліття описав Мирослав Антонович: «Український церковний спів перебрав, поєднав і розвинув різні первні тих музичних культур, що, з одного боку, мають, так би мовити, універсальні, а з другого – національні, питомо українські риси. Складний процес культурно-історичного ендосмосу між Україною і культурами Орієнту та Окциденту виявляв постійно тенденції розвитку від універсального до національного, а далі до реґіонального і льокального, самобутньо українського» (Антонович 1988–1989, 458). Весь цей процес відбувається в системі осьмо­ гласся, у якій визначена послідовність щотижневої зміни богослужбових текстів, піснеспівів тощо. Еволюція від монодичного одноголосся до партесного співу відбулася на початку XVII ст. Хор став звучати в багатоголосному складі від 3 до 48 голосів. У 1675 р. Микола Дилецький задля якісного виконання партесних творів написав «Граматику мусікійську», яка пояснювала основи професійного виконання багатоголосної музики в новітній ладовій системі. Разом із Дилецьким майстрами композиції партесного співу були Симеон Пекалицький, Герман Левицький, Іоанн Домарацький. Паралітургійні піснеспіви в ті часи існували нарівні з літургійними, однак не були занесені до системи осьмогласся та не були обов’язковими для відпусту канонічного чину. З XVI по XVIII ст. основними жанрами паралітургійної музики стають канти, псалми. Деякі з них згодом стають літургійними. Зародження та популяризація кантів зумовлені формуванням поезії книжною українською мовою здебільшого в містах, а саме в навчальних закладах: бурсах, академіях, братських школах тощо. В осередках освіти нарівні з іншими дисциплінами зазвичай викладали музичну грамоту, була поширена практика церковного співу. Канти мали світські (побутові, любовно-ліричні) і духовні (величальні Христу, Богородиці; покаянні) сюжети. Покаянні канти ідеологічно еволюціонували з епохи Бароко, перейнявши Олена Яремчук. Полістилістика паралітургійної музики Святослава Луньова  функцію моралізації християнської філософії, глибоко вкорінюючись у молитовну практику, подібно до монодії. Структурні елементи кантів еволюціонують від давньої традиції знаменної, або крюкової, нотації до класичних музичних форм через становлення сучасної ладово-тональної системи, тактової структуризації паралельно з канонічною музикою. Класичний період композиторської творчості, який характеризується обміном знаннями і композиторськими техніками з Європою, датують другою половиною XVIII ст. Найвпливовіші постаті, що сформували вектор розвитку професійної музичної спільноти та мали величезний вплив на композиторів-наступників, – це Микола Бортнянський, Артемій Ведель, Максим Березовський. Завдяки цій плеяді геніїв паралітургійна музика, що доповнювала церковну службу, стає унікальною самостійною одиницею. Відтепер композиції на канонічні тексти створюються для виконання не лише під час служби, а й для концертного вжитку, що означає використання набагато ширшої палітри музичних засобів – від виконавського складу до фактури, динаміки тощо. У XIX–XX ст. на обрій українського класичного простору виходять такі творчі постаті, як Микола Лисенко, Микола Леонтович, Яків Степовий, Кирило Стеценко, Олександр Кошиць. Це популяризатори та збирачі українського фольклору, які навчали любити рідну землю, її культуру, за що зазнавали репресій від більшовиків. Переслідування культурних і наукових діячів за часів Радянського Союзу зумовило підпільний спротив усіх мистецьких організацій, які все ж не дали українській культурі та освіті піти в небуття. Представники цього складного періоду – Григорій Верьовка, Левко Ревуцький, Борис Лятошинський, Ігор Шамо, Георгій Майборода та ін. На початку XXI ст. в Україні актуалізується процес духовного відродження. Відбувається розкриття свідчень про забуті, заборонені, замовчані події для широкого інформаційного простору. Велика частина репресій за часів Радянського Союзу була спрямована на церковну діяльність і місце автентичної духовної ієрархії як всередині держави, так і за її межами. Історичний шлях розвитку церковної культури, що на початку минулого століття був навмисне майже знищений, отримує можливість реабілітації як результат налагодження церковно-державних відносин. Нині духовна музика викликає неоднозначну оцінку серед науковців, філософів, композиторів, церковних діячів та вірян різних конфесій. 67 Церковний хоровий спів, що супроводжує богослужіння в храмах, часто характеризується як духовна музика. Проте твори сучасних композиторів на канонічні тексти церковного вжитку здаються деяким священнослужителям, парафіянам та навіть професійним музикантам недоречними під час богослужіння. Такій музиці сьогодні дають визначення «паралітургійна». Є багато різноманітних прикладів паралітургійної музики у творчості сучасних українських композиторів, як-от Леся Дичко, Валентин Сильвестров, Євген Станкович, Вікторія Польова, Олександр Козаренко, Віктор Степурко, Святослав Луньов та ін. Сьогодні паралітургійна музика виходить далеко за межі церковно-обрядових канонів загалом завдяки технікам написання, складу виконавців, інструментів, світловим ефектам тощо. Обираючи напрям паралітургії, композитор у такий спосіб виражає свою глибоку віру, створює власну неповторну інтерпретацію архаїчних розспівів. Вибір молитовних текстів, упорядкованих в унікальне полотно, є витонченим індивідуальним трактуванням глибинного розуміння канону. Самоідентифікація композитора відтворюється через накладання на канонічний текст різноманітних музичних і художніх засобів: звукових і світлових ефектів, неформальних для православного вжитку технік написання, крос-культурних нашарувань, сценічних дій тощо. Зацікавленість церковною тематикою у С. Луньова виникла в студентські роки під час бесід з його викладачкою теоретичних дисциплін у Національній музичній академії України та регентом за сумісництвом – Іриною Чижик. Композитор для своєї симфонії для мішаного хору a cappella обирає один із найскладніших і найстрашніших періодів для християн – Страсну седмицю – останній тиждень перед воскресінням Ісуса Христа, під час якого відбуваються такі події: зрада, засудження та страта Спасителя. На цей сюжет композитора надихнуло відвідання у 90-х роках XX ст. острова Валаам, зокрема руїн старого монастиря. За спогадами С. Луньова, острів повністю з каменю, навіть стовпи там не закопані, а присипані камінням. Після Другої світової війни Валаамський монастир перетворили на місце ліквідації та примусового працевлаштування інвалідів із заслань, концтаборів, тюрем. Радянське експансивне минуле, дитинство та виховання композитора вплинули на проєктування індивідуальних космологічних моделей. Святослав Луньов порівнює свою композиторську творчість з «ремонтом старих кораблів» (за І. Стравинським), що нагадує синтез роботи з музикою, різноманітними 68 ISSN 2617-8907. Наукові записки НаУКМА. Історія і теорія культури. 2023. Том 6 композиторськими техніками та засобами, подіями минулого. Це відображається в його симфонії для мішаного хору a cappella «Страсна седмиця», де відбувається нашарування людського болю, неосвіченості, розчарування на руїнах культового монастирського минулого. Симфонія або хорова симфонія як жанр – нова важлива ніша в системі сучасної вітчиз­ няної музики. Завдяки полістилістиці у твір вмонтовано ідеї та символи, які створюють багаторівневу історичну ретроспективу. У контексті паралітургійної музики в цьому творі, через його спрямування на виключно концертне виконання, відбувається переосмислення православних догм, реактуалізація найтрагічнішої події для християн. Основою музичного викладу матеріалу є знаменний розспів. Детально опрацьовувати старовинні наспіви С. Луньов мав змогу разом із В. Польовою в Іонинському монастирі, який композитор відвідував понад 20 років. Пропозиція реставрації обіходних піснеспівів надійшла від отця Дмитра Болгарського, диякона та регента. Працюючи зі старовинними наспівами, С. Луньов відкрив багато церковних сюжетів. У послідовності канонів та ірмосів, у текстах описано різні події, їхні наслідки та черговість. Вербальним першоджерелом хорової симфонії С. Луньова є тексти різних богослужінь, більшість з яких – це молитви, які використовуються лише під час Великого посту, а саме Страсного тижня. У цей період, згідно зі Святим Писанням, проходять останні дні земного життя Ісуса Христа. Хорова симфонія «Страсна седмиця» складається з семи частин. Важливо, що композитор влаштовує організацію їх послідовності не за канонічними правилами, а виокремлює найвагоміші канонічні тексти та інколи об’єднує декілька з них в одній частині. Першим номером С. Луньов обирає молитву «Отче наш». Як відомо, молитви «Отче наш» Ісус навчає своїх послідовників на їхнє прохання навчити молитися. Це головна молитва християнина, передбачає щоденне багаторазове індивідуальне промовляння віруючою людиною. У сучасній богослужбовій практиці ця молитва звучить на кожному богослужінні щонайменше тричі. Цей текст аналізували найвідоміші богослови, проте маємо неоднозначне, хоч і доволі розлоге трактування концепції побудови структури молитви. Гармонії, які композитор задіяв для передання вчення молитви Христа, – хиткі, гострі, створюють настрій глибокого відчуження від усього земного. Слова, які слугують зверненням до Господа, описують земне розуміння волевиявлення Творця: «Отче наш, іже єси на небесіх…», звучать монотонно, з глибоким натяком на розвиток подальшої трагедії. Другий номер «Нині сили небесния» містить у своєму складі вибрані рядки з трьох молитов: «Господи, воззвах к Тебі» (пс. 140), «Нині сили небесния» (співають замість «Херувимської пісні» на літургії Передосвячених Дарів), «Ангельський собор здивувався» (використано один вірш). Обʼєднано ці тексти тематизмом літургії Передосвячених Дарів, що в будні дні Великого посту замінює собою євхаристійну літургію та служиться в перші три дні Страсної седмиці. Літургія Передосвячених Дарів не містить прос­ комідії (частина, в якій з хліба та вина через молитви готується матеріал для причастя) та передбачає причастя вірян протягом тижня попередньо освяченими хлібом і вином у неділю на літургії св. Василія Великого. У складі літургії є 140-й псалом та «Нині сили небесния» (замість «Херувимської пісні»). Всі дії священництва під час «Нині сили небесния» відбуваються в повній тиші. А ось використання вірша «Благословен єси, Господи, научи мя…» зумовлене відсиланням до подій Великої суботи, коли риторика змінюється з покаянної на воскресну. Виклад тексту молитви «Нині сили…» підпорядкований звучанню слова. Такий спосіб музичного мислення, який відштовхується від вербального, характерний для знаменного розспіву та створює історичну ретроспективу з часом, коли ще не існувало тактової системи в церковній музиці. Текст молитви «Нині сили небесния» описує обʼєднане поклоніння як земного виміру, так і небесного добровільній жертві Ісуса Христа, який прийняв розпʼяття. Молитва закликає вірити глибині любові Бога до людей і через цю віру обіцяє вічне життя. Також на мелодії та ритмічній організації попереднього матеріалу «Нині сили…» звучать тексти віршів зі 140-го псалма. Текст 140-го псалма покаянний. Складається з віршів-прохань, які спрямовані до Господа задля захисту слів, дій та помислів від обману. Святослав Луньов використовує прийом виконання різних текстів на одну мелодію, поширений у церковній практиці співу тропарів, ірмосів, стихир тощо. Третій номер «Се Жених грядет» – молитва, яку співають на утрені перші три дні Страсного тижня. У тексті закладено відсилання до притч з Євангелія. Дві притчі пояснюють образ Жениха (Мв. 25:5-13; Лк. 12:36:38), інші описують останні повчання Ісуса (Мв. 21:19; Лк. 13:34-35; Мв. 21:23-24; Мв. 21:28-32; Мв. 21:33-42). Олена Яремчук. Полістилістика паралітургійної музики Святослава Луньова  Молитва оповідає про Ісуса, якого тут іменують Женихом, який може прийти в життя людини без попередження, в будь-який час. У тексті прописано настанови, як не пропустити зустріч зі Спасителем, – бути уважним, не присипляти свою совість, оскільки сон, у контексті молитви, рівносильний смерті та вигнанню з Царства Божого. Текст «Се Жених грядет в полунощі, і блажен раб, Єго же обрящет бдяща», що відкриває номер, завдяки фактурі викладу доноситься з різних теситурних вимірів, створюючи ефект реверберації та присутності в храмі. Весь номер загалом тонально не виходить за межі споріднених ладів, має чітку структуру, містить пряме відсилання до церковних обіходних піснеспівів. Четвертий номер «Єгда славнії ученици» – це тропар, що звучить перед причастям у Страсний тиждень. Текст оповідає про Таємну вечерю та учнів Христа, про Іуду, який вирішив зрадити свого вчителя. Номер, як і попередній, має стійку гармонію. Донесення тексту «Тогда Іуда омрачашеся» є одним із ключових моментів тлумачення людського гріха, який Ісус через розпʼяття спокутує. Пʼятий номер «Приідіте, ублажим Іосифа» – це стихира, яка виконується в Страсну пʼятницю під час цілування плащаниці Христа. Йосип Ариматейський – таємний послідовник Ісуса, був заможним чоловіком, радником синедріону, у час страти залишився з Марією, Никодимом та мироносицями біля розпʼяття. Після страти Ісуса Йосип іде до Понтія Пілата з проханням забрати тіло Христа та, домовившись, купує нову плащаницю, в яку згодом його загорнуть, а також віддає свою висічену в скелі гробницю, щоб там сховати тіло від вандалів. Музичний матеріал пʼятої частини стилізований під старовинні наспіви. У номері виписано соло тенора – образ Йосипа, та соло сопрано – образ Марії. Мелодія написана в східній стилізації, багата на мелізми та насичена драматичними інтонаціями. Шостий номер «Світе тихий» – наймас­ штабніший номер усієї симфонії. Основну молитву розділено на три частини, між ними виписано ще три молитви: «Да молчит всяка плоть», «Царю небесний» з відпустом, «Святий Боже». Всі молитви обʼєднує поховальна, траурна тематика. «Світе тихий» – це молитва, яка оповідає про подвиг Ісуса, про його добровільну страту заради людей; «Да молчит всяка плоть» – співають замість «Херувимської» у Велику суботу; «Царю небесний» – часто використовувана молитва, на панахидах і літіях (поховальні та поминальні обряди) переходить 69 у «Святий Боже». Цей номер містить пряму цитату грузинського гімну Богородиці «Ти є лоза виноградна», що символізує віру у воскресіння, в помилування. Останній, сьомий номер «Воскресеніє Твоє» – тропар пасхи, який констатує воскресіння Христа. Цей тропар, незалежно від наспіву, завжди звучить урочисто, життєствердно. Композитор імітує грузинський наспів, класично віддаючи початок чоловічим голосам: зазвичай на службах першими цей тропар починають співати священнослужителі, після чого їм вторять усі парафіяни. Використана класична форма виконання – тричі. Номер закінчується фрагментом молитви «Хресту Твоєму поклоняємось, Владико», який переходить у фінальні рядки православного піснеспіву «Воскресіння Христове бачачи», а саме: «распятіє бо притерпів, смертію смерть разруши». Фінал масштабний, урочистий, просвітлений. Висновки. Симфонія для мішаного хору a cappella «Страсна седмиця» С. Луньова створює ефект присутності під час найвагоміших і найтрагічніших подій у земному житті Ісуса Христа. Слухач, занурюючись у атмосферу, переживає разом з хором усі трансформації, перебуваючи у «звукових декораціях», побудованих на знаменному, грузинському, східному наспівах – найдавніших наспівах православної церкви. Матеріал викладено в різних фактурах, що калейдоскопічно репрезентують фрагменти еволюції церковної музики. Твір написаний виключно для концертного виконання, проте використання окремих частин під час служби вразило своєю глибиною та самобутністю, бо мелодика й мотиви побудовані в традиціях старовинних церковних наспівів. Духовні традиції, на яких побудований сюжет і безпосередній музичний базис аналізованого твору, разом із індивідуальними композиторськими методами, до яких вдається автор, перебувають у стані глибокої культурологічно-музичної взаємодії та репрезентують еволюцію мислення в царині інтерпретації духовних концепцій. Використання алгоритмічної техніки композиції, з переосмисленням технічних характеристик форми симфонії (за інтерпретацією композитора залишається лише її ідеологічна функція обʼєднання в спільне полотно), репрезентує національну, властиву українському народові масову хорову пісню, яка сягає корінням ще часів язичництва. Культурологія та музика уподібнюють багатогранний історичний та технологічний до­ свід, демонструючи багатошаровість загальної 70 ISSN 2617-8907. Наукові записки НаУКМА. Історія і теорія культури. 2023. Том 6 еволюції світу, ролі людини в ньому, та дають можливість загального аналізу напряму розвитку та наслідків цієї еволюції. Основною спільною ознакою для музики та культурології є тяжіння до цілісності, що зумовлено спільними джерелами духовного, прогрес і симбіоз яких простежено в хоровій симфонії «Страсна седмиця» Святослава Луньова. Список використаної літератури Александрова, Наталія. 2008. «Літургічна музика як жанрово-­ стильовий феномен у творчості вітчизняних композиторів кінця ХХ – початку ХХI століття». Автореф. дис. канд. мистецтвозн., Одеська державна музична академія ім. А. В. Нежданової. Антоненко, Маргарита. 2020. «Православна духовна музика в системі української культури кінця XX – початку XXI століття». 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Vasylchenko-Mikhno, Halyna. 1993. “Hretskyi rozspiv v ukrainskii spivochii praktytsi kintsia XVI – pershoi polovyny XVII st.: pro skhozhist z hreko-vizantiiskoiu himnohrafichnoiu tradytsiieiu.” Олена Яремчук. Полістилістика паралітургійної музики Святослава Луньова  PhD diss. abstract, Tchaikovsky Kyiv State Conservatory [in Ukrainian]. Volovchuk, Yuliia. 2018. “Symphony for mixed chorus “Holy Week” by Sviatoslav Lunev: author’s interpretation of spiritual and musical traditions.” Scientific Herald of Tchaikovsky National Music Academy of Ukraine 123: 49–57. https://doi. org/10.31318/2522-4190.2018.123.152417 [in Ukrainian]. Yasinovsky, Yuriy. 2015. “Musicology in Contemporary Scientific Discourse, or Can Music Studies Become a University Discipline in Contemporary Ukraine?” Naukovi Zapysky UCU [Annals of the UCU]. History Series 7 (2): 27–48. http://er.ucu. edu.ua/handle/1/658 [in Ukrainian]. 71 Yefimenko, Adelina. 2010. “Liturhichnyi zhanr yak interaktsiia: kontekst suchasnoi kompozytorskoi tvorchosti.” Scientific Herald of Tchaikovsky National Music Academy of Ukraine 85: 343–57 [in Ukrainian]. Zosim, Olha. 2011. “Bohosluzhbova muzyka u suchasnomu muzychnomu prostori Ukrainy : liturhichnyi aspekt.” Aktualni problemy istorii, teorii ta praktyky khudozhnoi kultury 26: 297–304. Kyiv: Milenium [in Ukrainian]. Zubko, Zhanna. 2011. “The geometry of canon (A. Pärt, S. Lunyov, M. Shalygin).” In Kyivske muzykoznavstvo. R. Glier Kyiv Institute of Music. https://www.lunyov.com/press/ [in Ukrainian]. Olena Yaremchuk THE POLYSTYLISTICS OF SVYATOSLAV LUNYOV’S PARALITURGICAL MUSIC Paraliturgical music is a sophisticated expression of the composer’s pure faith, acquired through knowledge of his craft in historical and technical progress. Many examples of sacred music have come down to us from the past, and most of them were in the territory of modern Ukraine, along with works by Russian composers that are still a priority in some churches. One way to rethink this topic is to study the work of Ukrainian composers, particularly in the context of paraliturgical music. One example is the work of contemporary Ukrainian composer Svyatoslav Lunyov. While many scholarly studies have been analyzing liturgical genres and music in the liturgy in various aspects, including canonical music and the work of S. Lunyov, the question of the place of the choral symphony “Strasna Sedmytsia” in the context of paraliturgical music remains unresolved. Due to its scientific novelty, the study of Lunyov’s choral symphony in the context of Ukrainian paraliturgical music became the subject of a special musical and cultural analysis for the first time. Since the beginning of the twenty-first century, the spiritual revival has become more and more important. The historical path of development of church culture, which was deliberately almost destroyed at the beginning of the last century, is being rehabilitated as a result of the establishment of church-state relations. Currently, sacred music is subject to very mixed opinions among scholars, composers, church leaders, philosophers, and people of different faiths. Music based on the canonical texts that form the basis of the church rite but interpreted by contemporary composers seems impossible to introduce into the service on an everyday level. Such music is defined as “paraliturgical.” Today, there are many different examples of music of this genre, as almost all academic composers turn to. Keywords: paraliturgical music, polystylistics, spiritual, Strasna Sedmytsia (Passion Week), prayer, ritual, Svyatoslav Lunyov. Матеріал надійшов 22.03.2023 Creative Commons Attribution 4.0 International License (CC BY 4.0) 

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Modern Scientific Research International Scientific Journal 2023 Vol1 Issue1 УДК 622.244.43-032.26 СТАБИЛИЗАЦИЯ РАСТВОРОВ ДИСПЕРСНЫХ СИСТЕМ ОБРАБОТАННЫМ БЕНТОНИТОМ МЕСТНОГО МЕСТОРОЖДЕНИЯ 1 М.А.Зияева1, С.Матсалаева2, А.Т.Есимбетов2. Ташкентский государственный технический университет Нукусский филиал Cамаркандского государственного университета ветеринарной медицины, животноводства и биотехнологии 2 Аннотация В статье приведены результаты фотометрических, рентгеноструктурных и реологических исследований растворов глин Навбахорской месторождений. Рентгеноструктурный анализ глин пропитанных новыми разработанными нами полимерами показывает, что в процессе модификации происходит частичное разрушение кристаллической структуры полигорскита, вследствие чего его базальтные отражения на рентгеновской дифрактограмме фиксируются частично. Ключевые слова: глина, месторождение, сорбция, ионы, буровой раствор, свойства, реология, твердый остаток, водоотдача, скважина. Abstract The results of photometric, X-ray diffraction and rheological studies of clay solutions of the Navbahor field are presented in the article. X-ray diffraction analysis of clays impregnated with new polymers developed by us shows that during the modification a partial destruction of the crystalline structure of polygorskite takes place, as a result of which its basaltic reflections on the X-ray diffractogram are partially fixed. Key words: clay, deposit, sorption, ions, drilling mud, properties, rheology, solid residue, water loss, well. Поиск и разведка месторождений нефти и газа в Республике Узбекистан на новых площадях и рост глубин бурения предопределили объективную необходимость в усовершенствовании технологии бурения нефтяных и https://academicsresearch.ru/index.php/MSRISJ 65 Modern Scientific Research International Scientific Journal 2023 Vol1 Issue1 газовых скважин, а также составов буровых растворов. Как известно, свойства буровых растворов, в первую очередь, зависят от химического состава воды и активных добавок к ним, из которых они приготовлены. Для получения на их основе качественных буровых растворов требуется специальная обработка большим количеством различных дефицитных и дорогостоящих активных химических реагентов. Для приготовления бурового раствора на водной основе необходим материал, создающий дисперсную фазу. Этим материалом является глина. Существует много разновидностей глин. Химический состав разнообразен, но общим является содержание окиси кремния (кремнезем) и окиси алюминия (глинозем), а также некоторое количество воды. Состав глины условно записывается: хА12О3.уSiО2.zН2О (водный алюмосиликат). Минералы глинистых пород: монтмориллонит, гидрослюда, палыгорскит, каолинит. Глинистые минералы состоят из мельчайших плоских кристалликовпластинок, между которыми проникают молекулы воды. Это и есть процесс распускания глины. При проводке 70% скважин в Устюрском регионе, широко применяются недиспергирующие системы буровых растворов, содержащие полимерную композицию селективного флокулянта и стабилизатора в сочетании с высококачественным бентонитом. Флокулянт (сополимеры винилацетата и малеинового ангидрида, частично гидролизованный полиакриламид ГПАА, сополимеры акрилата натрия и метилметакрилата, полиоксиэтилен и прочие) предотвращает диспергирование частиц выбуренной породы и облегчает удаление их из раствора на системе очистки, а также регулирует структурно-механические свойства раствора. Стабилизатор (полиакрилат натрия ПАН) «обволакивает» дисперсные частицы бентонита, снижая показатель фильтрации раствора и не вызывая изменения его вязкости. Кроме того, полиакрилат натрия защищает бентонитовую фазу раствора от присутствующего в системе флокулянта. https://academicsresearch.ru/index.php/MSRISJ 66 Modern Scientific Research International Scientific Journal 2023 Vol1 Issue1 Недиспергирующий раствор считается качественным, если отношение частиц выбуренной породы в твёрдой фазе к эквивалентному содержанию в ней бентонита составляет примерно 1:1. Содержание глинистой твёрдой фазы в данной системе не должно превышать 4% об. В процессе углубления скважины высокомолекулярный флокулянт (ГПАА) минимизирует наработку коллоидной глинистой фазы, поступающей в буровой раствор [1,2]. В результате концентрация коллоидной глинистой фракции (с размером частиц менее 1 мкм) становится катастрофически низкой. Помимо флокулирующего действия, присутствующего в системе полиакриламида, обеднение бурового раствора коллоидной глинистой фракцией наступает также в результате первичной коагуляции при поступлении минерализованных пластовых вод, кислых газов (углекислоты) или вследствие загрязнения бурового раствора частицами выбуренной породы (зашламование). В то же время для поддержания требуемого уровня структурных характеристик, эффективной удерживающей способности бурового раствора концентрация коллоидной фазы в недиспергирующем растворе должна быть не менее 25 – 30 кг/м3. Причём более 70% дисперсных частиц (слоистые пластинки) должны иметь размер менее 1 мкм и нормальное распределение поверхностного электрического заряда – «грани» заряжены отрицательно, а «рёбра» – положительно. Именно в этом случае обеспечивается «здоровая» коагуляционная структура глинистого раствора типа «грань-ребро» («карточный домик»), создающая на стенках скважины непроницаемую плотную фильтрационную корку и характеризующаяся оптимальными выносящей и гелеобразующей характеристиками. При снижении же концентрации коллоидной фазы менее 20 кг/м 3 структура недиспергирующего бурового раствора разрушается (величина СНС стремится к нулю). Для поддержания необходимых структурных характеристик в этом случае промывочная жидкость должна обрабатываться https://academicsresearch.ru/index.php/MSRISJ 67 Modern Scientific Research International Scientific Journal 2023 Vol1 Issue1 ксантановым биополимером (до 2 кг/м3), что повышает стоимость бурового раствора. К тому же биополимер быстро теряет эффективность в результате целого ряда факторов: биологическое разложение, механодеструкция молекул, сорбция на частицах выбуренной породы и удаление реагента из раствора. Установлено, что поддержание необходимых структурно-механических характеристик недиспергирующего раствора эффективно достигается обработкой циркулирующего бурового раствора 4–6%-ной бентонитовой суспензией, приготовленной из высококоллоидального бентонита. В результате введения бентонитовой суспензии концентрация микронной коллоидной фазы в недиспергирующем буровом растворе не должна снижаться менее 25 – 30 кг/м3. В качестве высококоллоидального бентонита оптимально использование обработанного со стабилизатором ГСБ-1 бентонита Навбахорского месторождения. В состав глин Навбахарского месторождения входят монтмориллонит (ММТ), полигорскит, кальцит и гидрослюда. Структура ММТ Al4Si8O20(OH)4∙nH2O. У смешанно слойных минералах кристаллическая решетка построена из нескольких упорядоченных прослоенных решеток (пакетов) значительной толщины и соответствует сумме высот составляющих слоев. Один кристаллит ММТ может иметь разные по свойствам поверхности: две на внешней стороне и одна внутри кристалла. Внешними могут быть базальтные поверхности и поверхности скола. Внутренние базальтные поверхности отдельных слоев, т.е. чистая ММТ структура, состоит из слоя кремнекислородных тетраэдров, заключенного между двумя слоями алюмокислородно - гидроксильных октаэдров. ММТ способен впитывать воду в межпакетные пространства и значительно набухать. Проведенные исследованиями выявлено, что разработанные полимерные композиции, по существу, быстро диспергирующегося в воде концентрата бурового раствора и стабильных полимерных суспензий, которые могут быть https://academicsresearch.ru/index.php/MSRISJ 68 Modern Scientific Research International Scientific Journal 2023 Vol1 Issue1 высушены для получения таких концентратов. Нами экспериментально было найдено оптимальное соотношение между растворимой в воде и нерастворимой составляющими для данного физико-химического состава реагента-стабилизатора. Такой концентрат может быть легко вновь диспергирован в воде. Отсутствие слипаемости способных к слипанию и нерастворимых в воде частиц делает такой концентрат легко вновь диспергируемым в водной среде, что еще раз подчеркивает превосходства разработанных нами полимерных стабилизаторов на основе отходов и местных сырьевых ресурсов стабилизаторов буровых растворов. ЛИТЕРАТУРА 1. Ангелопуло О.К., Подгорнов В.М., Аваков В.Э. Буровые растворы для осложненных условий. – М.: Недра, 1988. – 135 с. 2. Улитин М.В., Филиппов Д.В., Лукин М.В. Физико-химические свойства, устойчивость и коагуляция лиофобных дисперсных систем. – ГОУ ВПО Иван. гос. химико-технол. ун-т.- 2007.- С. 74 – 81. 3. Холмберг К., Йенссон Б., Кронберг Б., Линдман Б. Поверхностноактивные вещества и полимеры в водных растворах.- М.: Бином.- 2007.- С. 178 – 185. 4. Казицына Л.А., Куплетская Н.Б. Применение УФ-,ИК-, ЯМР- и Массспектроскопии в органической химии. –М;Издат.МГУ. 2009 г. - 237 с. https://academicsresearch.ru/index.php/MSRISJ 69 

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HEALTH INEQUALITIES AND WELFARE STATE REGIMES: THEORETICAL INSIGHTS ON A PUBLIC HEALTH ‘PUZZLE’ Clare Bambra Address for correspondence: Professor Clare Bambra, Department of Geography, Wolfson Research Institute, Queen's Campus, Durham University, Stockton on Tees, TS17 6BH. Tel: 0191 3340417, Fax: 0191 3340361, clare.bambra@durham.ac.uk The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non-exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in Journal of Epidemiology and Community Health and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence (http://jech.bmj.com/ifora/licence.pdf). Article word count = 2644 1 ABSTRACT Welfare states are important determinants of health. Comparative social epidemiology has almost invariably concluded that population health is enhanced by the relatively generous and universal welfare provision of the Scandinavian countries. However, most international studies of socioeconomic inequalities in health have thrown up something of a public health ‘puzzle’ as the Scandinavian welfare states do not, as would generally be expected, have the smallest health inequalities. This essay outlines and interrogates this ‘puzzle’ by drawing upon existing theories of health inequalities - artefact, selection, cultural-behavioural, materialist, psychosocial, and life course to generate some theoretical insights. It discusses the limits of these theories in respect to crossnational research; it questions the focus and normative paradigm underpinning contemporary comparative health inequalities research; and it considers the future of comparative social epidemiology. 130 words 2 BACKGROUND It is now widely acknowledged that welfare states are important determinants of health as they mediate the social determinants of health.[1] Welfare state provision varies extensively, but typologies have been put forward to categorise them into distinctive types - welfare state regimes. [1][2][3] Welfare state regimes have increasingly been used within social epidemiology to analyse crossnational differences in population health. [4][5][6][7][8] These studies have almost invariably concluded that population health is enhanced by the relatively generous and universal welfare provision of the Social Democratic Scandinavian countries, especially when contrasted to the AngloSaxon welfare states.[4][5][6][7][8] The different types of welfare state and their constituent countries are described in Box 1. However, in contrast to their comparatively strong performance in terms of overall health, data from most – but not all - of the recent comparative studies of health inequalities in the general population suggest that the Scandinavian welfare states do not have the smallest health inequalities. [9][10][11][12] For example, Mackenbach et al’s Europe wide study of inequalities in mortality found “no evidence for systematically smaller inequalities in health in countries in northern Europe (Scandinavia)”.[9] Indeed, relative inequalities in mortality were smaller in the Southern (Italy, Spain, Portugal) and Bismarckian (Netherlands, Belgium, Germany, France) countries.[9] Data is provided from three other example studies of health inequalities in Europe in Table 1.[10][12] Given the higher levels of social expenditure in the Scandinavian welfare states, the smaller income inequalities, and the commitment to equality underpinning the Social Democratic welfare model in Scandinavia, it has long been something of a ‘puzzle’ in public health as to why the Scandinavian countries do not have the smallest health inequalities.[13][14][15] This essay draws upon the theories of health inequalities to scrutinise this ‘puzzle’.[16][17][18][19][20] THEORETICAL INSIGHTS ON COMPARATIVE HEALTH INEQUALITIES Box 2 outlines the main theories of health inequalities. [16][17][18][19][20] These are commonly used to explain socio-economic health inequalities within countries. In this paper they are applied to crossnational differences in the magnitude of socio-economic health inequalities and used to offer insights into the ‘puzzle’ as to why health inequalities are not the smallest in the Scandinavian welfare states. 3 Artefact The artefact explanation questions the existence of health inequalities, considering them to be a mere artefact of data collection and measurement (Box 2). Applying it to the issue of comparative health inequalities, leads to the conclusion that the ‘public health puzzle’ - of why health inequalities are not the smallest in the Scandinavian countries - is not in fact a real puzzle, but simply the result of the data and methods used. Certainly, the application of different indicators of social inequality (e.g. income, occupation and education) and the use of different datasets has produced divergent results (see Table 1). Different cross-national patterns also emerge in terms of the different ways in which specific indicators of inequality are calculated. For example, studies of educational inequalities can compare those with average years of education to those with one standard deviation below the national average, [21] or the difference between those with no education or only primary education compared to those with tertiary education (Box 2).[22] There are also more general issues in terms of making cross-national comparisons of health inequalities as it is not clear whether the bottom groups are the same in each country and whether their composition changes over time.[21][23] The use of relative or absolute measures of health inequalities is also an important issue (see Discussion section). There is of course another clear measurement problem which is the use of ‘welfare state regimes’, a concept which assumes a homogenous approach to welfare provision within and between the countries of any particular regime type.[1][24] Health Selection The health selection approach asserts that health determines socio-economic class status rather than socio-economic class determining health (Box 2). This would imply that the social consequences of ill health would need to be greater in the Scandinavian countries and that people who have ill health are more likely to be concentrated in the lower socio-economic groups. Instinctively, such direct selection seems unlikely given the extensive employment protection for people with ill health within the Nordic countries and their comparatively high replacement rates for people out of the labour market due to sickness or disability.[18] Selection is also considered to be more influential in respect to income related inequalities than educational ones and so it is unlikely to explain the results of the comparative studies of educational inequalities in health.[24] 4 Culture and Behaviour The cultural-behavioural approach asserts that the link between socio-economic class and health is a result of differences between socio-economic classes in terms of their health-related behaviour (Box 2). In terms of physical activity and diet, there is no evidence of larger inequalities in the Scandinavian countries, at least as measured by educational inequalities in obesity.[9] However, socio-economic inequalities in smoking are much higher in the Nordic countries than in the other welfare state regimes.[9][14] Similarly, inequalities in deaths from cardiovascular disease are higher in the Scandinavian countries (except Denmark) as compared to other European countries.[9] This, it is argued, is because the Scandinavian countries are at a very mature stage of the smoking epidemic with the majority of smoking behaviour concentrated in the least educated groups.[25] This suggests that one consequence of the Social Democratic welfare states is that the universal health messages and health promotion interventions are taken up by the middle classes first.[14] This results in what has been referred to as ‘intervention generated inequalities’, as whilst the health of everyone improves, that of the middle classes does so at a faster rate.[26] Materialist Explanations The (neo)materialist explanation focuses on income and what income enables such as access to goods and services and the limitation of exposures to physical, and psychosocial, risk factors (Box 2). Applying a materialist perspective may initially seem somewhat limited as the Scandinavian countries have the smallest income inequalities and offer largely universal welfare services.[27] However, as Diderichsen has commented, lower levels of income inequality do not negate inequalities in exposure to the other material determinants of health.[28] Further, as has consistently been shown, social inequalities in access to services remain even within universal systems e.g. the inverse care law in relation to nationalised health services.[29][30] There is certainly tentative evidence to suggest that inequalities in total avoidable mortality (as a result of diseases amenable to medical intervention) are higher in the Scandinavian countries than elsewhere.[31] From a slightly different angle, there have been long standing criticisms that the Social Democratic welfare states operate on an insider/outsider 5 basis with vulnerable ‘outsider’ groups, such as immigrants, often marginalised and without entitlement to the full benefits of the universalist system.[32] Psychosocial Psychosocial explanations focus on the biological and health consequences of how social inequality makes people feel (Box 2). From a psychosocial perspective then, it has been speculated that ‘relative deprivation’ may be a factor behind the larger than expected relative health inequalities in the Scandinavian welfare states.[14] Relative deprivation will occur in all unequal societies, including the Nordic welfare states. Following Dahl and colleagues, it is possible to speculate that the effects of relative deprivation may be more extensive in the Nordic welfare states because of the high levels of expectation of upward social mobility and prosperity that they generate amongst the less privileged, expectations that are seldom met.[14][15][33] This may increase health inequalities especially in stress related conditions, such as heart disease, or indeed self-assessed health.[33] Life course Life course epidemiology has highlighted how different causal mechanisms and processes may lie behind the social gradient in different diseases (Box 2).[16] This may also be the case in terms of the inequalities in different welfare state regimes. For example, a study found that in both Britain and Sweden, lone mothers were more likely to report poor health than couple mothers.[34] However, the pathways leading to the health disadvantage of lone mothers were very different in the two countries: poverty and worklessness were the primary issues in Britain, but not in Sweden.[34] Extrapolating from this example, it is possible to suggest that the same outcomes - socio-economic health inequalities - may be present in all welfare state regimes to a greater or lesser extent, but as a result of different causal mechanisms. This suggests that the welfare state regimes approach is perhaps too generalised and only able to offer a rough guide to inequalities.[35] DISCUSSION These theoretical insights are rather limited and somewhat speculative: none of the theories alone can provide a wholly convincing explanation. Whilst there appears to be some power to the cultural- 6 behavioural perspective, really, beyond issues of artefact, it is very difficult to explain why health inequalities are not smaller in the Scandinavian countries through reference to existing theories of health inequalities. This is perhaps because all the other theories (selection, psychosocial, materialist, life course) to a greater or lesser extent expect health inequalities to be smaller in the Scandinavian countries. This may indicate that the existing theoretical explanations are lacking and need to be combined and developed. Certainly, no single theory is able to empirically explain within country inequalities, never mind between country ones. Alternatively, of course, it may be that the contrasting performances of the Scandinavian welfare states in regards to overall health versus health inequalities cannot really be considered to be a ‘puzzle’ at all. Firstly, there have only been a small number of cross-national comparative studies conducted to date and these have focused on the health gap rather than the social gradient.[9][10][11][12] Secondly, the use of welfare state typologies has been extensively critiqued not least on the grounds that it obscures important policy differences between welfare states (e.g. the flexicurity of Denmark compared to the protectionism of Sweden or Norway).[35][36] Further, some have argued that there is a need to move beyond Scandinavian welfare state exceptionalism and to acknowledge the commonalities that there are between, say, the Bismarckian and Scandinavian models, particularly in terms of the status of the lowest socio-economic groups; as well as the progress of other welfare states, such as Japan, in terms of creating healthy environments.[37] This suggests that comparative social epidemiology should shift focus and conduct comparisons of more precise policy areas and specific social determinants (such as the work environment) instead.[38][39] This could enable a deeper and more nuanced understanding of how particular national policies, or the shared policies of specific welfare state regimes, impact on health inequalities.[35] Another factor that needs to be taken into consideration is that the ‘puzzle’ has emerged partly as a result of the focus of comparative epidemiological research on relative, as opposed to absolute, measures of health and inequality. This has meant that the Scandinavian countries are effectively victims of their own success, as whilst they have substantially improved the health of all, the high level of health of the middle classes has meant that relative social inequalities remain.[35] This, it could be argued, is the real issue in terms of why the Scandinavian countries perform comparatively poorly in 7 terms of relative health inequalities, and, as Lundberg (2008) has pointed out, this is an achievement, not something to be criticised.[35] The lowest socio-economic groups in the Scandinavian countries are objectively better off in absolute terms than the lowest socio-economic groups in the other welfare state regimes. For example, the absolute mortality risk difference between manual and non-manual is lowest in Sweden and Norway.[40] There is also emerging evidence to suggest that amongst the most vulnerable social groups - the old, the sick, and children - there are smaller socio-economic inequalities in the Social Democratic welfare states.[41][42][43] Indeed, there is by no means an accepted research consensus that relative health inequalities amongst the general population are not the smallest in the Nordic countries as, for example, Borrell and colleagues’ analysis of data from individual country health interview surveys suggested that the Social Democratic countries did exhibit the smallest adult health inequalities.[44] Further, it has been shown that relative measures of inequalities are negatively associated with total population health: countries with lower overall mortality tend to experience larger inequalities in mortality.[45] This is perhaps because the social determinants of population health differ from the determinants of health inequalities.[46] The use of absolute or relative measures of health inequality also raises important normative and political issues about whether the role of the welfare state is to improve the status of those at the very bottom of society or whether it is about promoting general equality. Implicitly, cross-national research to date has tended to favour the latter view, however, it is possible to suggest that it should move beyond relative comparisons and focus instead on absolute ones. This would perhaps also enhance the policy relevance of such research,[47] after all, as Geoffrey Rose famously commented, “relative risk is not what decision-taking requires … relative risk is only for researchers; decisions call for absolute measures”.[47] Future comparative research could therefore benefit from examining the absolute health of the most marginalised, poorest and vulnerable within different types of welfare state. The limits of the study of the formal welfare state are also perhaps exposed by the ‘puzzle’. Comparative social epidemiology has to date largely focused on analysing the influence on health and health inequalities of the formal and the public – the state, the economy, politics, public policies, welfare services and social benefits. In contrast, there has been relatively little attention paid to the 8 potential influence on differences in cross-national health and health inequalities of the informal and the private side of welfare capitalism - unpaid care, the family, community and social support, and different constructions of gender roles.[48][49][50] For example, some studies have suggested that those countries with a higher proportion of unpaid family care and domestic labour by women have smaller health inequalities.[50] Such social differences in the informal welfare sector could therefore be a factor behind the smaller than expected health inequalities found by some studies in the Southern and Bismarckian, welfare states.[50][51] The impact of the social - the private and the informal welfare sphere - on comparative health inequalities is under-explored in public health and might provide important insights. However, as Raphael and Bryant’s research has noted, women’s health is more sensitive to public welfare and is improved by high levels of state social welfare, [52] so Bartley’s assertion that analysing the social sphere is challenging and complex is therefore well made.[50] The intersectional nature of inequality – gender, social class and ethnic stratifications – is therefore also something that needs to be considered in future cross-national research on health.[53] CONCLUSION The existence, extent, interpretation and causes of the Scandinavian public health ‘puzzle’ remain controversial. On the one hand, the ‘puzzle’ highlights the limitations of existing theories of health inequalities and thereby challenges conventional public health thinking. On the other hand, it has been seen to act as a distraction away from the real potential of comparative social epidemiology in providing detailed assessments of the public policies of different welfare states and how the social determinants vary. However, the issue of the ‘puzzle’ highlights the strong, and often unacknowledged, normative tensions within comparative social epidemiology in terms of whether the welfare state is about creating overall equality, or improving the situation of the poorest and most vulnerable, or both. The future of comparative social epidemiology research will be largely determined by the shifting balance of power in this debate both in terms of the empirical research agenda and the extent of theoretical evolution. The latter may well benefit from an increased interaction with social policy, social theory and political economy perspectives.[54] 9 ACKNOWLEDGEMENTS Earlier versions of this paper were presented at the Hertie School of Governance, Berlin; Department of Social Medicine, University of Copenhagen; Department of Sociology, University of Ghent; NOVA, Oslo; Department of Society and Globalisation, Roskilde University; Department of Geography, St Andrews University; and the Centre for Health Equity Studies, University of Stockholm. I am grateful for the informative feedback I received at these events. COMPETING INTEREST None declared 10 What is already known on this subject Population health is enhanced by the relatively generous and universal welfare provision of the Scandinavian countries. However, some international studies of socio-economic inequalities in health have thrown up a public health ‘puzzle’ as the Scandinavian welfare states do not, as would generally be expected, have the smallest health inequalities. What this study adds This paper outlines and interrogates this ‘puzzle’ by drawing upon existing theories of health inequalities - artefact, selection, cultural-behavioural, materialist, psychosocial, and life course. It finds that these theories provide little insight into the issue, and that whilst this may be a result of poor theory development in public health, it may also demonstrate the limitations – both methodological and conceptual - of contemporary comparative social epidemiology. Policy Implications The paper raises normative issues about whether the role of the welfare state and public health policy is about improving the status of those at the very bottom of society (absolute measures of health) or about promoting general equality (relative measures of health). A focus on the absolute health of the most vulnerable as well as an awareness of the social sphere and intersectionality could enhance the policy relevance of comparative health research. 11 REFERENCES [1] Bambra, C. (2007), 'Going Beyond The Three Worlds of Welfare Capitalism: Regime theory and public health research ', Journal of Epidemiology and Community Health, 61, 1098-1102. [2] Ferrera, M. (1996). The southern model of welfare in social Europe. Journal of European Social Policy, 6, 17-37 [3] Esping-Andersen, G. (1990). The Three Worlds of Welfare Capitalism. London: Polity [4] Navarro, V., Muntaner, C., Borrell, C. et al. (2006). Politics and Health Outcomes. 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Journal of Epidemiology and Community Health, 63: 867-868. 15 Table 1: Summary findings from three example comparative studies of socio-economic inequalities in self-reported health (bad/poor versus fair/good/very good) by welfare state regime Study Eikemo et al [12] Measure of inequality Education average education versus one sd below average Summary of Results* Men Absolute Prevalence Rate Difference Relative Prevalence OR (95% CI) Absolute Prevalence Rate Difference Relative Prevalence OR (95% CI) 6.4 1.19 (1.14-1.24) 5.7 1.25 (1.20-1.30) 9.6 1.35 (1.23 – 1.48) 8.2 1.29 (1.18-1.41) 10.5 1.44 (1.35-1.53) 12.1 1.54 (1.44-1.64) 17.3 1.69 (1.58-1.81) Bismarckian Anglo-Saxon Scandinavian Southern 14.8 Eikemo et al [10] Income – top versus bottom income tertiles Bismarckian 1.68 (1.50 – 1.89) 11.6 1.81 (1.62 – 2.03) 10.9 1.79 (1.46 – 2.19) 14.8 2.14 (1.77 – 2.57) 13.0 1.97 (1.70 – 2.27) 15.8 2.14 (1.84 – 2.49) 17.4 2.86 (2.12 – 3.70) 17.4 2.73 (2.17 – 3.44) 11.2 1.24 (1.12-1.37) 12.7 1.31 (1.19-1.45) 13.3 1.43 (1.26-1.63) 13.7 1.36 (1.21-1.52) 18.9 1.87 (1.45-2.42) 24.2 1.75 (1.39-2.21) Southern Anglo-Saxon Social Class (Education aspects = secondary or more versus less than secondary) Christian Democratic Social Democratic Late Democracies 1.57 (1.47-1.69) 9.8 Scandinavian Espelt et al [11] Women * Age standardised differences between the top and bottom socio-economic groups in each analysis 16 Box 1: Welfare state regimes [1] Liberal/residual In the welfare states of the liberal regime (UK, USA, Ireland, Canada, Australia), state provision of welfare is minimal, social transfers are modest and often attract strict entitlement criteria; and recipients are usually means-tested and stigmatised. In this model, the dominance of the market is encouraged both passively, by guaranteeing only a minimum, and actively, by subsidising private welfare schemes. The liberal welfare state regime thereby minimises the decommodification effects of the welfare state and a stark division exists between those, largely the poor, who rely on state aid and those who are able to afford private provision. Conservative/Corporatist/Bismarckian The conservative welfare state regime (Germany, France, Austria, Belgium, Italy and, to a lesser extent, the Netherlands) is distinguished by its ‘status differentiating’ welfare programs in which benefits are often earnings related, administered through the employer; and geared towards maintaining existing social patterns. The role of the family is also emphasised and the redistributive impact is minimal. However, the role of the market is marginalised. Social Democratic/Scandinavian The Social Democratic regime type (Nordic countries), is characterised by universalism, comparatively generous social transfers, a commitment to full employment and income protection; and a strongly interventionist state. The state is used to promote social equality through a redistributive social security system. Unlike the other welfare state regimes, the Social Democratic regime type promotes an equality of the highest standards, not an equality of minimal needs and it provides highly decommodifying programs. Southern/Latin It has been proposed that the Southern European welfare states (Italy, Greece, Portugal and Spain) comprise a distinctive, southern, welfare state regime. The southern welfare states are described as ‘rudimentary’ because they are characterised by their fragmented system of welfare provision which consists of diverse income maintenance schemes that range from the meagre to the generous and welfare services, particularly, the health care system, that provide only limited and partial coverage. Reliance on the family and voluntary sector is also a prominent feature. 17 Box 2: Theories of Health Inequalities [16][17][18][19][20] Artefact The artefact approach suggests that socio-economic inequalities do not really exist but are a result of the data used and methods of measurement: that difference in health by socio-economic class can be explained by differences in measurement and that the size of the inequalities observed is due to differences in data measurement tools. Health Selection The health selection approach asserts that health determines socio-economic class status rather than socio-economic class determining health. Individuals who are ‘fitter’ are more likely to move up the social hierarchy. In contrast, people with ill health are downwardly mobile (or less upwardly mobile) and are therefore concentrated within the lower socio-economic classes. Cultural-Behavioural The cultural-behavioural approach asserts that the link between socio-economic class and health is a result of differences between socio-economic class in terms of their health related behaviour: smoking rates, alcohol and drug consumption, dietary intake, physical activity levels, risky sexual behaviour, and health service usage. Such differences in health behaviour, it is argued, are themselves a consequence of disadvantage and unhealthy behaviours may be more culturally acceptable amongst lower socio-economic class. Materialist The (neo)materialist explanation focuses on income and what income enables such as access to goods and services and the limitation of exposures to physical, and psychosocial, risk factors. Materialist approaches give primacy to structure in their explanation of health and health inequalities, looking beyond individual level factors (agency), in favour of the role of public policy and services such as schools, transport and welfare in the social patterning of inequality. Psychosocial Psychosocial explanations focus on how social inequality makes people feel and their biological and health consequences. Social inequality leads to long term feelings of subordination or inferiority which in turn stimulate chronic stress responses which have profound consequences for physical and mental health. The socio-economic class gradient is therefore explained by the unequal social and economic distribution of psychosocial risk factors. Life Course The life course approach combines aspects of the other explanations, thereby allowing different causal mechanisms and processes, to explain the social gradient in different diseases. Health inequality between socio-economic classes is the result of inequalities in the accumulation of social, psychological, and biological advantages and disadvantages over time. 18 

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REVIEW published: 26 October 2020 doi: 10.3389/fcvm.2020.598400 Vascular Manifestations of COVID-19 – Thromboembolism and Microvascular Dysfunction Kirsty A. Roberts 1† , Liam Colley 2† , Thomas A. Agbaedeng 3 , Georgina M. Ellison-Hughes 4* and Mark D. Ross 5* 1 Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, United Kingdom, 2 School of Sport, Health & Exercise Science, Bangor University, Bangor, United Kingdom, 3 Centre for Heart Rhythm Disorders, School of Medicine, The University of Adelaide, Adelaide, SA, Australia, 4 Centre for Human and Physiological Sciences, Faculty of Life Sciences & Medicine, School of Basic and Medical Biosciences, King’s College London, London, United Kingdom, 5 School of Applied Sciences, Edinburgh Napier University, Edinburgh, United Kingdom Edited by: Andrew F. James, University of Bristol, United Kingdom Reviewed by: Jian Xu, University of Oklahoma Health Sciences Center, United States Katsuya Hirano, Kagawa University, Japan *Correspondence: Georgina M. Ellison-Hughes georgina.ellison@kcl.ac.uk Mark D. Ross m.ross@napier.ac.uk † These authors have contributed equally to this work and share first authorship Specialty section: This article was submitted to Atherosclerosis and Vascular Medicine, a section of the journal Frontiers in Cardiovascular Medicine Received: 24 August 2020 Accepted: 28 September 2020 Published: 26 October 2020 Citation: Roberts KA, Colley L, Agbaedeng TA, Ellison-Hughes GM and Ross MD (2020) Vascular Manifestations of COVID-19 – Thromboembolism and Microvascular Dysfunction. Front. Cardiovasc. Med. 7:598400. doi: 10.3389/fcvm.2020.598400 The coronavirus pandemic has reportedly infected over 31.5 million individuals and caused over 970,000 deaths worldwide (as of 22nd Sept 2020). This novel coronavirus, officially named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), although primarily causes significant respiratory distress, can have significant deleterious effects on the cardiovascular system. Severe cases of the virus frequently result in respiratory distress requiring mechanical ventilation, often seen, but not confined to, individuals with pre-existing hypertension and cardiovascular disease, potentially due to the fact that the virus can enter the circulation via the lung alveoli. Here the virus can directly infect vascular tissues, via TMPRSS2 spike glycoprotein priming, thereby facilitating ACE-2-mediated viral entry. Clinical manifestations, such as vasculitis, have been detected in a number of vascular beds (e.g., lungs, heart, and kidneys), with thromboembolism being observed in patients suffering from severe coronavirus disease (COVID-19), suggesting the virus perturbs the vasculature, leading to vascular dysfunction. Activation of endothelial cells via the immune-mediated inflammatory response and viral infection of either endothelial cells or cells involved in endothelial homeostasis, are some of the multifaceted mechanisms potentially involved in the pathogenesis of vascular dysfunction within COVID-19 patients. In this review, we examine the evidence of vascular manifestations of SARS-CoV-2, the potential mechanism(s) of entry into vascular tissue and the contribution of endothelial cell dysfunction and cellular crosstalk in this vascular tropism of SARS-CoV-2. Moreover, we discuss the current evidence on hypercoagulability and how it relates to increased microvascular thromboembolic complications in COVID-19. Keywords: COVID-19, endothelium, pericyte, coronavirus, thromboembolism INTRODUCTION In January 2020, the Center for Disease Control recognized a new coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is believed to have originated from the Wuhan city in Hubei province, China. As of the 22nd September 2020, over 31.5 million people worldwide have been infected, with currently over 970,000 deaths recorded (1). According Frontiers in Cardiovascular Medicine | www.frontiersin.org 1 October 2020 | Volume 7 | Article 598400 Roberts et al. Vascular Manifestations of COVID-19 The initial evidence of the cardiovascular impact of COVID19 was provided in cross-sectional cohort studies which observed significantly elevated hs-cTnI and hs-cTnT levels, suggestive of myocardial injury in these patients (14, 18, 19). High levels of these cardiac biomarkers are related to worse prognosis of the disease (19, 20), with a number of studies demonstrating a higher risk of admission to ICU (10), requirement for mechanical ventilation (12), and incidence of arrhythmias and death from COVID-19 (3, 4, 10, 12, 19) in those with elevated circulating hscTnI or hs-cTnT levels. Moreover, the mortality risk associated with elevated hs-TnI/T was greater than that observed for advanced age, pre-existing diabetes, respiratory disorders, and CAD (10, 12). The elevations in hs-TnI/T are also associated with elevated levels of NT-ProBNP and C-reactive protein (CRP), suggesting the myocardial injury observed in COVID19 patients may be linked with ventricular dysfunction and inflammation (12). There are several potential reasons for the elevated cardiac injury observed in COVID-19 patients with worsening outcomes. These include direct viral infection of the myocardium, the use of anti-viral medications (18), the side-effects of the COVID-19 associated cytokine storm (21), or likely a combination of the three. Viral entry is likely, as SARS-CoV-2 is known to enter human cells via binding of the transmembrane protein, the angiotensin-converting enzyme 2 (ACE2) receptor, which is highly expressed in both the lungs and the heart (22). In fact, due to this mechanism of entry, there has been debate on the use and potential benefit of the use of ACE inhibitors in patients with cardiac injury and/or hypertension (23), with the American Heart Association, The Heart Failure Society of America, and the American College of Cardiology publishing a joint consensus statement for the treatment of COVID-19 patients with ACE inhibitors (24). Cardiovascular events, such as incidences of acute coronary syndrome (ACS) or acute myocardial infarction (AMI) in COVID-19 patients have been demonstrated (25), indicating that the impact of COVID-19 on the cardiovascular system leads to cardiovascular-related mortality. The root causes of COVID-19 ACS/AMI remain unknown, but could be due to the elevated myocardial demand as a result of the infection, akin to type 2 MI, cytokine-induced atherosclerotic plaque instability and rupture, or non-plaque thrombosis (25–27). Although, as documented, there is a clear impact of the virus on the myocardium, either directly or indirectly; however, the potential role of the vasculature in COVID-19 associated cardiovascular complications has been relatively overlooked, and may be prognostically important in these patients. In fact, in a recent study by Chen et al. (28) using a single cell atlas of the human myocardium showed that ACE2 is expressed on pericytes in the heart (28), suggesting that viral infection of pericytes, which surround the endothelial lining of blood vessels, could lead to microvascular inflammation in the heart tissue, resulting in non-obstructive MI. Therefore, the following sections will investigate the impact of COVID-19 on vascular tissues, specifically endothelial cells and pericytes, and the to the World Health Organization (WHO) the total case fatality rate (CFR) is 3.1%, but this varies significantly depending on geographical location. For example, the USA have a CFR of 2.9% (6,740,464 cases), whereas the United Kingdom and Italy have significantly higher CFRs of 10.6% (394,261 cases) and 12.0% (298,156 cases), respectively (1). The SARS-CoV2 infection gives rise to COVID-19 disease, which typically results in fever, respiratory distress (shortness of breath and cough) (2–4), and subsequent respiratory failure. Symptoms often arise between 2 and 14 days after infection (5), and the risk of mortality due to COVID-19 appears greater in older individuals (6), and in individuals with comorbidities, such as hypertension (7), coronary artery disease (CAD), and diabetes mellitus. Despite patients reporting with symptoms relating to fever and respiratory distress, there is growing evidence for the involvement of the cardiovascular system. Patients often exhibit elevated cardiac biomarkers such as cardiac troponin I/T (hs-cTnI/hs-cTnT) (3, 4, 6, 8–11) and N-terminal pro-Btype natriuretic peptide (NT-proBNP) levels (8, 12), which suggest myocardial damage and ventricular/atrial dysfunction. However, the impact of COVID-19 on the vasculature is largely unknown, but there are case reports of viral infection of the endothelium (13), as well as elevated markers of coagulation, such as D-dimer in COVID-19 patients (14), which itself may indicate a significant risk of pulmonary thromboembolism (PTE) in patients. The focus of this review is to detail the effects of SARS-CoV2 and COVID-19 disease on the vasculature, whilst discussing the potential direct and indirect mechanisms which lead to endothelial damage and dysfunction. Moreover, we also discuss the pathogenesis of COVID-19 associated thromboembolism and its consequences upon the cardiovascular system and COVID-19 disease progression. EPIDEMIOLOGY OF COVID-19 AND CARDIOVASCULAR RISK Patient cohort studies show that there is a large prevalence of patients with COVID-19 who have comorbidities, such as hypertension (17–57% of all patients) and cardiovascular disease (CVD) (11–21% of all patients) (3, 15–17). Patients with hypertension or CAD are not only at greater risk of infection, and admission to hospital, but having one or more of these comorbidities also appears to increase the risk of progression of the disease (15). In a Chinese cohort, it was observed that in COVID-19 patients, 30% of them had hypertension (14). In the non-survivors, the incidence of hypertension was greater than that of survivors (48 vs. 23% of patients), and this was even more pronounced for incident coronary heart disease (24 vs. 1% of patients) (14). Hypertension and pre-existing CVD were also more common comorbidities in patients requiring admission to the intensive care unit (ICU) (18). Frontiers in Cardiovascular Medicine | www.frontiersin.org 2 October 2020 | Volume 7 | Article 598400 Roberts et al. Vascular Manifestations of COVID-19 subsequent involvement of these tissues on thrombotic risk in COVID-19. cells and endothelialitis within multiple tissue beds in patients with COVID-19. Although limited by a small sample size, the findings of Varga et al. (13) are supported by Ackermann et al. (40), who reported severe endothelial injury, viral infection, and disrupted cell membranes in seven lungs obtained post-mortem from individuals who died from COVID-19. When compared to seven lungs from individuals who died from influenza, microthrombi were nine times as prevalent in the lungs from the COVID-19 individuals. Furthermore, widespread microthrombi was accompanied by microangiopathy and occlusion of alveolar capillaries (40), which is in line with other studies (41), and can predispose organs to microinfarcts (42). An unexpected finding was the observation of intussusceptive angiogenesis, in which the degree was associated with the duration of hospitalization (40). Intussusceptive angiogenesis is the formation of new vessels, via non-sprouting angiogenesis, and is constructed of an endotheliallined “pillar” spanning the vessel lumen, which significantly alters the microcirculation (43). Cytoplasmic vacuolisation and cell detachment in pulmonary arteries (44), in addition to pulmonary capillary injury featuring neutrophil infiltration and fibrin deposition (41, 45) has also been reported, further demonstrating local endothelial cell perturbations within lung tissue. Moreover, renal post-mortem histopathological analysis by Su et al. (46) found endothelial cell swelling with foamy degeneration in 19% of patients, with 12% demonstrating a few areas of segmental fibrin thrombus in glomerular capillary loops that is associated with severe endothelial injury. Considering endothelial dysfunction leads to impaired systemic microvascular function, it seems likely that involvement of the vascular system’s first line of defense (endothelial cells) precipitates and propagates the systemic damage observed in severe cases of COVID-19, through altered vascular integrity, vascular inflammation, and via disruption of coagulation and inflammatory pathways (13, 33). The mechanisms for this have not yet been fully elucidated and are varied due to the heterogenic nature in which the virus affects individuals. Cardiometabolic comorbidities associated with poorer prognosis in COVID-19 patients have a strong association with pre-existing endothelial dysfunction (i.e., hypertension and CAD) (47, 48). It is therefore evident that understanding the role of endothelial cells in SARSCoV-2 infection is crucial to identifying potential therapeutic strategies to combat the virus and improve patient outcomes. The role of endothelial cells and potential mechanisms of endothelial cell dysfunction in COVID-19 are depicted in Figure 1. COVID-19 AND ENDOTHELIAL CELL DYSFUNCTION Initial SARS-CoV-2 infection occurs within the lung epithelia, whereby serine proteases, most notably transmembrane protease serine 2 (TMPRSS2), cathepsin B, and cathepsin L1, prime the SARS-CoV-2 spike glycoprotein, which is followed by ACE2-mediated viral entry (29). Infection of lung alveoli allows SARS-CoV-2 to enter the systemic circulation, subsequently predisposing multiple organs to potential infection. Coexpression of both key serine proteases and ACE2 is required for successful infection of cells by SARS-CoV-2 (29). Multiple organs contain cells which co-express ACE2 and these serine proteases, including the lungs, heart, kidneys, liver, and the vasculature (30–32). Microvascular dysfunction and the role of the vascular endothelium is increasingly implicated in the acute respiratory distress syndrome (ARDS) and systemic impact of SARS-CoV2 infection. Endothelial cells protect the cardiovascular system and are crucial in regulating vascular homeostasis, preventing coagulation, controlling blood flow, and regulating oxidative stress and inflammatory reactions (33, 34). There is growing evidence of a vascular involvement in the pathogenesis of severe COVID-19, with imaging studies revealing perfusion abnormalities within the brains of patients with COVID-19 presenting with neurological issues (35), in addition to perfusion abnormalities within the lungs of COVID-19 pneumonia patients (36). Moreover, cross-sectional studies have reported a high incidence of coagulopathies, characterized by elevated Ddimer and fibrinogen concentrations, which lead to thrombotic events and are associated with poor outcomes (37, 38), thus demonstrating the potential involvement of endothelial cells in the pathophysiological consequences of COVID-19. Endothelial Cell Involvement in COVID-19 Involvement of endothelial cells in the pathophysiology of COVID-19 goes beyond coagulation derangements, with SARSCoV-2 being shown to directly infect engineered human blood vessel organoids and human kidney organoids in vitro (39). This has been confirmed, in vivo, by histological studies demonstrating viral infiltration into endothelial cells, with Varga et al. (13) reporting endothelial cell involvement across multiple organs (e.g., lungs, heart, intestines, kidneys, and liver) in three patients; two of whom died (multisystem organ failure; myocardial infarction, and subsequent cardiac arrest, respectively) and one survived. Viral infection of endothelial cells was observed in a transplanted kidney of one patient with evidence of endothelial cell inflammation (endothelialitis) within cardiac, small bowel, lung, and liver tissue of two patients. Furthermore, one other patient demonstrated endothelialitis of the submucosal vessels within the small intestine, which was accompanied by a reduced left ventricular ejection fraction. These findings demonstrate direct viral infection of endothelial Frontiers in Cardiovascular Medicine | www.frontiersin.org Potential Mechanisms of Endothelial Dysfunction in COVID-19 Angiotensin-Converting Enzyme 2 (ACE2) ACE2 is an endogenous negative regulator of the reninangiotensin system (RAS) and has been identified as the key receptor facilitating viral entry of SARS-COV-2 (49, 50), along with key serine proteases to prime the spike glycoprotein of the virus, most notably TMPRSS2 (29), which is expressed by endothelial cells (30). ACE2 is widely expressed in cells throughout the body, from the respiratory tree to the vascular 3 October 2020 | Volume 7 | Article 598400 Roberts et al. Vascular Manifestations of COVID-19 FIGURE 1 | The role of endothelial cells and mechanisms of endothelial cell dysfunction in COVID-19. (A) SARS-CoV-2 infects endothelial cells through angiotensin-converting enzyme 2 (ACE2) mediated viral entry, facilitated by TMPRSS2 priming the SARS-CoV-2 spike glycoprotein. Infection of endothelial cells may result in a downregulation of ACE2, promoting an imbalance between ACE2 and angiotensin II (AngII) levels, in favor of AngII. Moreover, infection of either endothelial cells or pericytes will perturb the crosstalk between these two cells, thus contributing to endothelial cell dysfunction. (B) In severe cases of COVID-19, activated macrophages release various cytokines (e.g., soluble interleukin 2-receptor [IL-2R], interleukin-6 [IL-6] and tumor necrosis factors [TNFs]), which are attributed to the exaggerated immune-mediated cytokine storm and can result in vascular inflammation (endothelialitis) as a result of increased adhesion molecule expression on endothelial cells and inter-endothelial gaps, thus promoting vascular hyperpermeability. Activated endothelial cells can contribute to the cytokine storm by releasing various cytokines in response to damage and dysfunction, contributing to a vicious cycle of inflammation and oxidative stress that inhibits the release of vasoactive factors (e.g., nitric oxide [NO]), thus favoring vasoconstriction and further contributing to vascular permeability. Abnormal activation of platelets and endothelial cells is the key process leading to thrombosis, which represents the role of endothelial cell dysfunction in the pathogenesis of thromboembolism in COVID-19 patients. Subsequently, the dislodgement of thrombotic clots creates a mobile embolus that disseminates intravenously, thereby leading to thromboembolic complications in COVID-19. A downregulation in the expression of ACE2, as a result of viral entry into cells, disrupts the regulation balance between angiotensin II (Ang II) and ACE2, indirectly affecting the vasculature. This imbalance facilitates an elevation in the expression of Ang II, subsequently promoting an atherogenic state across the cardiovascular system, especially inflammation and oxidative stress, whilst also elevating blood pressure by stimulating an increase in sympathetic nervous system system, heart, kidneys, liver, gut, central nervous system, and retina, and is recognized as eliciting protective effects, particularly against CVD (49). The expression of ACE2 in many organs allows relatively easy transport of the virus throughout the body (51). Consequently, interference of the physiological processes associated with ACE2 by viral entry of SARS-CoV2 is likely to explain the multi-organ dysfunction pertaining to endothelial cells that is seen in severe cases of COVID-19. Frontiers in Cardiovascular Medicine | www.frontiersin.org 4 October 2020 | Volume 7 | Article 598400 Roberts et al. Vascular Manifestations of COVID-19 this cytokine storm (65–67), leading to a loss of vascular barrier integrity and likely promoting pulmonary oedema, thereby causing endothelialitis, and activation of coagulation pathways. Cross-sectional studies have consistently demonstrated marked elevations in pro-inflammatory markers, such as soluble interleukin-2 receptor (IL-2R), interleukin-6 (IL-6), CRP, and tumor necrosis factors (TNF) (6, 12, 68). This marked elevation in pro-inflammatory markers has been linked with mortality and promotes inter-endothelial gaps and thus vascular hyperpermeability (69, 70), along with exacerbating oxidative stress. IL-6 in particular is associated with increased vascular permeability, a hallmark of the inflammatory response (71, 72), and IL-6 levels are directly correlated with the severity and mortality of COVID-19 (14, 73, 74). Moreover, IL-6, along with other cytokines released from activated macrophages, such as IL1β, activate endothelial cells via elevations in adhesion molecules (42) leading to a myriad of vascular disturbances including leukocyte tethering to the vascular bed, platelet aggregation and coagulation derangements. activity (52). This is supported by studies reporting marked elevations in plasma AngII concentrations in patients with COVID-19 (53) and also being linked to disease severity in patients infected with novel influenza A (54). This pathophysiological increase in Ang II and without the modulator and protective effects of Ang 1-7, results in downstream elevation of plasminogen activator inhibitor-1 (PAI-1) from endothelial cells, further accelerating vascular inflammation and the facilitation of the coagulation cascade (42), thus resulting in endothelial damage (55). Elevated PAI-1 is a hallmark of endothelial dysfunction, promoting increases in circulating endothelial microvesicles, resulting from endothelial shedding via activated cells, which pose a risk of thromboembolic events (56, 57). Some have argued that following cell entry of SARSCoV-2, down-regulation of ACE2 receptors may result in an indirect activation of the kallikrein-bradykinin pathway, thereby promoting an increase in vascular permeability and thus leading to oedema and microcirculatory dysfunction (33, 58, 59). It has been suggested that kinin inhibition may be a potential therapeutic approach to reducing vascular leakage into the lung, and therefore, oedema (60). Kinin inhibition may, therefore, promote endothelial repair through reducing vascular permeability, although whether this is an effective therapeutic approach is yet to be confirmed within the literature. In contrast to this, consistent reports of hypokalaemia in patients with severe COVID-19 (61, 62) suggest an increase in aldosterone, via elevations in Ang II, resulting in an increase in ACE, which acts to metabolize bradykinin (63). Therefore, the role of bradykinin in the pathogenesis of microvascular dysfunction in COVID-19 is questionable and more likely a result of the effects of Ang II, stemming from a downregulation of ACE2 after viral entry into cells. Moreover, given that hypokalaemia is associated with ventricular arrhythmias that are commonly observed in COVID19 (18), it is plausible that this is a contributing mechanism to both endothelial dysfunction and arrhythmogenesis. Oxidative Stress An overproduction of reactive oxygen species (ROS) in infected cells is a key factor in viral replication of respiratory viruses and subsequent tissue damage (75). Following viral infection, endothelial activation and regulation of adhesion molecules leads to neutrophil activation, which results in the production of a plethora of histotoxic mediators including ROS (59). This has implications for the onset and progression of the cytokine storm since, as described above, endothelial cells are key orchestrators of cytokine overload. The ensuing oxidative stress, defined as a systemic imbalance between ROS (or free radicals) and antioxidants, causes an increased expression of prothrombotic and cell-surface adhesion molecules (76). Oxidative stress may therefore be linked to the pathogenesis and severity of COVID-19 infections (77) and peri-endothelial ROS production in COVID-19 may, therefore, contribute to the multi-organ failure associated with severe disease, which seems likely given that it has previously been demonstrated in the pathogenesis of other viral infections, such as SARSCoV and influenza (78, 79), and ARDS (80). The elevation in ROS accumulation promotes oxidative stress and nuclear factor kappa B (NF-κB) signaling, with the potential for dysregulated antioxidant mechanisms, such as Nrf2 and antioxidant response element signaling, promoting the release of various endothelial genes, such as endothelin and adhesion molecules, thus favoring vasoconstriction and increased vascular permeability (81, 82). The elevation in free radical production, potentially as a combined result of increased Ang II expression, proinflammatory responses, and a reduced capacity for free radical scavenging by impaired antioxidant signaling, impairs endothelial function. Elevated superoxide concentrations, promoted by the release of mitochondrial-derived ROS is a hallmark of oxidative stress, which facilitates the quenching of nitric oxide (NO) and the formation of the secondary free radical, peroxynitrite, in turn reducing NO bioavailability (83). Moreover, this process uncouples endothelial The Cytokine Storm The mechanisms involved in the pathogenesis of microvascular dysfunction in COVID-19 patients, although not yet fully understood, are likely not solely attributed to direct viral infection of endothelial cells. Endocytosis or membrane fusion of SARS-CoV-2 to cells either leads to cell damage or apoptosis which activates the immune response and the release of various cytokines promoting an exaggerated inflammatory environment (42). Moreover, endothelial cells regulate local and systemic inflammatory reactions and immune responses (33) and activation of these cells via the exaggerated immune-mediated inflammatory response of SARS-CoV-2 may present an indirect mechanism of endothelial damage and dysfunction among the COVID-19 patient population. Endothelial cells produce various cytokines and chemokines and have been identified as central regulators of an exaggerated systemic inflammatory response, or “cytokine storm” (64), a common feature of severe SARS-CoV-2 infection (65). More severe cases of COVID-19 are associated with progressive lung damage which has, in part, been attributed to Frontiers in Cardiovascular Medicine | www.frontiersin.org 5 October 2020 | Volume 7 | Article 598400 Roberts et al. Vascular Manifestations of COVID-19 score” (90). DIC has been linked with multi-organ system failure within the COVID-19 population (38, 91, 92), demonstrating a pro-coagulant state of the vasculature. Furthermore, mild thrombocytopenia can be found in 70 to 95% of patients with severe COVID-19, however, it has not been found to be an important predictor of outcome (21, 93). Therefore, the presence of coagulopathy within patients with COVID19 should be considered as an endotheliopathy, rather than traditional DIC. nitric oxide synthase, which further elevates superoxide production, contributing to the pro-oxidant environment of the vasculature. Such elevations in oxidative stress would promote antioxidant signaling, however, numerous respiratory viral infections, such as respiratory syncytial virus, human metapneumovirus, and influenza, have perturbed antioxidant defense mechanisms by inhibiting antioxidant enzyme induction (84). Interestingly, it has been proposed that Nrf2 activators could be a potential therapeutic strategy for inhibiting viral entry of SARS-CoV-2 (85), and may also pose a benefit to endothelial repair and functioning by the scavenging of free radicals, reducing oxidative stress, and inhibiting pro-inflammatory signaling. Cellular Cross-Talk: Endothelial Cells and Pericytes Pericytes share a basement membrane with endothelial cells, which is formed, maintained, and remodeled successfully through cellular cross-talk between these two cells, demonstrating that pericytes and endothelial cells have an extensive linkage and are key for maintaining basement membrane, and thus vascular barrier integrity. This has been confirmed by cell-to-cell interaction analysis, demonstrating that endothelial cells are the main cross-talking cell with pericytes within cardiac tissue, with a predominant role of angiopoietin ligands (ANGPT1/2) and Tie receptor 2 (TIE2) maintaining endothelial cell stability and function in capillary vessels (28). A balance between ANGPTs and TIE2 is key for the maintenance of endothelial stability and vascular integrity (28, 94); therefore, it is possible that a breakdown of the cross-talk between pericytes and endothelial cells disrupts this balance and results in a compromised vasculature that is prone to a pro-inflammatory, pro-coagulant state. Whilst these findings were observed in normal heart tissue, this is supported by a pericyte-specific infection by SARS-CoV-2 in experimental (95) and human histological studies (96). Whilst there is evidence of a direct viral infection of endothelial cells, some have argued that endothelial cell dysfunction is a result of pericyte infection. Cardot-Leccia et al. (96) reported wall thickening of the venules and alveolar capillaries in lung tissue of a deceased COVID19 patient, accompanied by a marked decrease in pericytes, compared to normal lung parenchyma. Combined with the findings of He et al. (95) and the highly infectious potential of pericytes demonstrated by single cell RNA sequencing studies (28), these data seem to support a potential “pericyte hypothesis” as a mechanism for microvascular dysfunction in the pathogenesis of COVID-19. Moreover, infection and loss of pericytes would result in a dysregulation of the cross-talk between pericytes and endothelial cells, promoting capillary endothelial dysfunction, which would explain the wall thickening of venules and capillaries observed in the data from Cardot-Leccia et al. (96). Taken together, pericytes seem to have the potential as a highly infectious cell population for SARS-CoV-2 and may contribute to endothelial dysfunction by promoting an imbalance between ANGPT1/2 and TIE2, perturbing vascular barrier integrity and increasing vascular permeability. However, the notion that it is solely pericytes that are infected and induce endothelial dysfunction is unlikely considering the compelling histological data presented within the literature (13, 40). Coagulation Cascade Perturbations to the endothelium may result in vascular leakage and promote inflammation, but also predispose the vasculature to a pro-coagulant state. Indeed, a common manifestation in patients with COVID-19 is the presence of coagulation abnormalities and instances of thromboembolism, which has been associated with disease severity and a higher incidence of mortality (38), whilst also increasing the risk of MI and stroke. The endothelium plays an important role in the prevention of thromboembolic events by regulating the coagulation cascade, achieved, in part, via inhibition of various tissue factors by a Kunitz-type protease inhibitor, known as the tissue factor pathway inhibitor (TFPI) that resides on the endothelial cell surface (34). The transmembrane protein tissue factor is required for in vivo coagulation by the binding and activation of various tissue factors (i.e., activation of factor Xa) promoting prothrombin conversion to thrombin, and thus the conversion of fibrinogen to fibrin (34, 86), inhibiting TFPI and promoting clot formation. TFPI is predominantly bound to the microvasculature (87), however, it has been demonstrated to play a role in the regulation of arterial thrombosis in mice (86). Marked coagulation derangements have been reported in a single-center cross-sectional study by Goshua et al. (88) who assessed markers of endothelial cell and platelet activation, namely circulating von Willebrand factor (vWF), soluble P-selectin and soluble thrombomodulin, in critically and non-critically ill COVID-19 patients. They observed that endotheliopathy is present in COVID-19 and is associated with increased mortality, with a suggestion that soluble thrombomodulin concentrations may predict mortality and clinical outcomes in COVID-19 patients. It was suggested that the coagulopathy observed in their data was distinctly separate from disseminated intravascular coagulation (DIC) and should be considered an endotheliopathy (88). The notion of a “COVID19 coagulopathy” is supported by a number of other studies. DIC has been reported to be characteristic of COVID-19, however, its presentation is different to that regularly observed in sepsisinduced DIC. In sepsis-induced DIC, marked thrombocytopenia is observed with a mild elevation in D-dimer concentrations (89), which is in contrast to DIC observed in COVID-19 patients (90). This is supported by only 14.7% (22 of 150) of patients scoring positive on the “sepsis-induced coagulopathy Frontiers in Cardiovascular Medicine | www.frontiersin.org 6 October 2020 | Volume 7 | Article 598400 Roberts et al. Vascular Manifestations of COVID-19 COVID-19 AND THE COAGULATION CASCADE - RISK OF THROMBOEMBOLIC EVENTS (NIHSS) was associated with 23% increased risk of COVID-19 positivity. Interestingly, in a retrospective multicentre study of stroke patients (107), 28% were later diagnosed with COVID19. However, the true burden of thromboembolism COVID-19 remains unknown and will, hopefully, be answered by larger prospective studies. There is evidence to suggest increased risk of thrombotic complications and stroke (both are hereafter referred to as thromboembolism for simplicity) in COVID-19 (97). At the mechanistic level, both venous and arterial thrombosis have been attributed to activation of inflammation and hypoxia, platelet activation, endothelial dysfunction, and circulatory stasis. However, the impact of thromboembolic complications on the prognosis of COVID-19, clinical course of thromboembolic disorders in these patients, and the impact of prophylactic and therapeutic anticoagulation therapies in COVID-19 are not well-known. Impact of Thromboembolic Complications on COVID-19 Prognosis The presence of underlying or incident thromboembolic complications is associated with poor prognosis of COVID-19. A history of thromboembolism is reported in 2.3 to 22% of severe cases compared to 0 to 6% in non-severe cases (108). Patients with prior neurologic thromboembolic complications are shown to have a 2.5-fold increased risk of COVID-19 severity (108) and D-dimer is often elevated above reference range in hospitalized cases (17). These patients are usually older, have a higher number of comorbidities, have a higher prevalence of ARDS, and are more likely to be non-invasively ventilated (109). Data also shows that patients with more severe COVID-19 have higher incidence rates of thromboembolic complications. For instance, 31% of patients admitted to the ICU developed thromboembolic complications during follow-up in one Dutch study (110). Yearly increment in age and prior coagulopathy, defined as prothrombin time >3 s or activated partial thromboplastin time (aPPT) >5 s, are shown as independent predictors of incident thromboembolic complications in severe COVID-19 (110). Diagnosis of pulmonary thromboembolism in ICU patients with COVID-19 is more common (at 21%) compared to 7% admitted due to influenza or 6% for all ICU patients (111). Additionally, the association between a history of thromboembolic complications and mortality has been analyzed in COVID-19 patients. The burden of underlying coagulopathy was reported in 50% of non-survivors in the Wuhan cases (14), with a D-dimer >1,000 ng/mL (reference range ≤250 ng/mL) shown to be an independent predictor of 18-fold greater risk of in-hospital mortality (14). A multicentre cohort from the US showed that the coagulation component of the SOFA score is associated with 64% greater odds of 28-day in-hospital death in a multivariable adjusted model (112). These observations are further supported by the results of a meta-analysis (113), which show a 2.4-fold elevated risk of mortality in COVID-19 patients with cerebrovascular disease, defined as stroke and brain infarction. Overall, these data highlight the risk, and subsequent poor prognosis of thromboembolism in COVID-19. Epidemiological Burden of Thromboembolism in COVID-19 The prevalence of neurologic manifestations, including cerebrovascular diseases, was reported at 36.4% in an earlier retrospective case series from Wuhan, China (98). In patients presenting with confirmed or suspected COVID-19, thromboembolism is prevalent at 20.4% (99). In the same study, six of the patients with laboratory findings demonstrated elevated D-dimer levels (>7,000 mg/L) and 40% of the patients had pulmonary thromboembolism. Another series showed that 67% of thromboembolic complications are ischaemic in origin, while 33% are haemorrhagic (100). In the pediatric population, thromboembolic complications are not common. For instance, elevation of D-dimer was not found in children with SARSCoV-2 compared to other inflammatory multisystem syndromes (101), and no thromboembolic event was found in children and adolescents in a large, multicentre European cohort (102). In addition to a prior history of stroke, patients with COVID-19 develop incident thromboembolism. The incidence rates of acute thromboembolic complications are reported between 5 and 32.5% in retrospective cohorts (103, 104). Underlying cardiovascular risk factors, including diabetes, hypertension, and a history of CVD, are implicated as univariate correlates (103). D-dimer levels at hospital admission are also significantly correlated with incident thromboembolism, with a negative predictive value of more than 90% (104). In a prospective cohort of 150 French COVID-19 patients vs. a historic cohort of 233 non-COVID-19 controls, COVID-19 ARDS independently predicted thromboembolic complications and pulmonary thromboembolism even after propensity score matching (90). The comorbid nature of thromboembolic lesions in patients with COVID-19 underscores some underlying predisposition to SARS-CoV-2 infection. Indeed, thromboembolic complications have been associated with depressed immune function and increased post-stroke infections. Infection rates ranging from 18.7 to 43.7% have been reported in patients with intracerebral hemorrhage (105, 106), with respiratory infections predicting almost six-fold higher risk of future thromboembolism (106). A 1-unit increment in National Institutes of Health Stroke Scale Frontiers in Cardiovascular Medicine | www.frontiersin.org Coagulation Cascades and the Mechanisms of Thrombosis in COVID-19 While significant associations have been noted for thromboembolism and SARS-CoV-2 infection and worsening of COVID-19, a causal relationship is not well-defined. However, there are data to suggest some mechanistic underpinnings (Figure 2). Laboratory investigations have demonstrated significant elevations of markers of coagulation cascades, such as D-dimer, aPPT, fibrinogen, and factor VIII. Ddimer ≥2,600 ng/mL and failure of clot lysis at 30 min on 7 October 2020 | Volume 7 | Article 598400 Roberts et al. Vascular Manifestations of COVID-19 of SARS-CoV-2 RNA in platelets of COVID-19 patients, which were shown to be hyperactivated and aggregated at a lower threshold of in vitro thrombin stimulation (125). Platelets from COVID-19 degranulate, which correlates with reduced platelet factor 4 and serotonin levels, and release extracellular vesicles to participate in coagulation (125). Consequently, platelet reprogramming could facilitate the transmission of SARSCoV-2 and promote thrombo-inflammation. Indeed, thromboinflammation mediated by distinct patterns of platelet and neutrophil activations, neutrophil-platelet aggregate formation, and neutrophil extracellular traps has been reported in COVID19 pneumonia (126). thromboelastography predicted future thromboembolic events in ICU patients with c-statistic of 0.78 and 0.74, respectively (114). This highlights the fact that shutdown of fibrinolysis occurs in COVID-19. In addition to coagulation markers, endothelial dysfunction may underlie the increased risk of thromboembolism in COVID-19 as both vWF activity and vWF antigen are increased in COVID-19 ARDS compared to non-COVID-19 ARDS (90). Thromboembolic complications might also be precipitated by underlying cardiovascular injury. For example, patients with co-existing ST-elevation MI and COVID-19 have significantly increased rates of thromboembolic complications, affecting multiple vessels and stents, thrombus grade post-percutaneous coronary intervention (115). Additionally, cardiac arrhythmias play an important role in the development of thromboembolic events, due in part to the shared underlying myocardial substrate (116). Cardiomyopathy, consisting of mechanical dysfunction, structural remodeling, and electrophysiological changes, is a common cause of both intracardiac thrombus and cardiac arrhythmogenic substrate formation (116). The presence of right-heart echodensity on transoesophageal and transthoracic echocardiography has been reported in COVID-19 patients (117–119). Interestingly, intracardiac thrombus coexisted with persistent tachycardia, global hypokinesis, left ventricular dysfunction, and right ventricular dilatation and reduced systolic function (117–119). Taken together, this indicates that thromboembolism in COVID-19 might be mediated via cardiacspecific pathologies. At the mechanistic level, thromboembolic complications may arise due to activation of inflammation and hypoxia, platelet activation, endothelial dysfunction, and circulatory stasis in COVID-19. Inflammatory overdrive and hypoxia may induce abnormalities of coagulation, the third component of the Virchow triad. On necropsy, areas of diffuse and extensive inflammatory infiltrations have detectable thromboemboli and microemboli (120). Direct infection of immune cells with SARSCoV led to activation of monocyte-macrophage differentiation, coagulation pathway upregulation, and increased cytokine production (121). SARS-CoV-2 might drive thromboembolic mechanisms by its utilization of the ACE-2 receptor, which is needed to clear Ang II from the circulation. Increased Ang II could, in turn, drive the release of vWF from endothelial cells and platelet activation via involvement of Na+ /H+ exchanger (122). Finally, the presence of auto-antibodies, such as lupus anticoagulant, might drive activated coagulation pathways and thromboembolic risk (123). Direct activation of platelets by SARS-CoV-2 is a likely pathway for the development of thromboembolism. Hottz et al. (124) reported platelet activation and formation of platelet-monocyte aggregates in patients with severe but not in mild COVID-19. Similar findings were observed when platelets from COVID-19 negative patients were treated with plasma from COVID-19 positive patients (124). Platelets from COVID-19 patients induces ex vivo expression of tissue factor (TF) in monocytes (124), indicating a likely reprogramming event during SARS-CoV-2 infection. Indeed, this hypothesis is supported by pre-publication evidence reporting the presence Frontiers in Cardiovascular Medicine | www.frontiersin.org Prophylaxis and Management of Thromboembolism in COVID-19 Given the high burden of comorbidities and mortality in patients with thromboembolic complications, proper and adequate anticoagulation is highly warranted. Current management of patients with severe COVID-19 includes subcutaneous low molecular weight heparin (LMWH), suspicion of venous thromboembolism in those with high D-dimer levels and rapid respiratory deterioration, and consideration of therapeutic anticoagulation in those in whom diagnostic testing is not possible and there is no apparent bleeding risk (127, 128). A retrospective series showed no mortality benefit with LMWH compared to non-users (129). However, in those with a high sepsis-induced coagulopathy score and markedly elevated Ddimer level, 28-day mortality was lower among users (129). There is also consideration of experimental interventions, such as plasma exchange or administration of anti-inflammatory drugs, in clinical trial settings. Nevertheless, there are several unknowns with the management of thromboembolism and associated complications in COVID-19. For instance, will prophylactic as compared to therapeutic anticoagulation result in a better outcome in these patients? A prospective cohort recently demonstrated significant reduction in pro-coagulants 7 days after thromboprophylaxis (130). However, the study was very limited by sample size. In another study, patients on prophylactic anticoagulation had higher venous thromboembolism than the therapeutic anticoagulant arm, although the latter group had a higher overall incidence of thromboembolic events, including pulmonary embolism (131). It is envisaged that these issues will be answered in ongoing clinical trials, such as the COVID-19 HD, a randomized controlled trial comparing high-dose vs. low-dose LMWH (132). SUMMARY In addition to the known impact on the respiratory system, emerging evidence strongly implicates COVID-19 as a vascular disease. Patients with pre-existing cardiovascular conditions which are commonly characterized by endothelial dysfunction are particularly at risk of downstream complications and COVID-19-associated mortality. Endothelial cell dysfunction, inflammation, and damage are implicated as a consequence 8 October 2020 | Volume 7 | Article 598400 Roberts et al. Vascular Manifestations of COVID-19 FIGURE 2 | The development and consequences of thromboembolism in COVID-19. The thromboembolic implications of SARS-CoV-2 are best conceptualized in three key stages. First, lung infection of SARS-CoV-2 can spill over, with a consequent cardiovascular tropism of the virus. Within the vascular beds, the increased level of Ang II, which occurs due to SARS-CoV-2 mediated depletion of ACE2, could drive the dysfunction of endothelial cells. This, and other independent pathways (i.e., direct infection of endothelial cells), could lead to the release of von Willebrand factors (vWF), which can activate circulating platelets via adhesive glycoprotein receptors (i.e., gpIb). Activated platelets form aggregates with monocytes and neutrophils, leading to enhanced production of pro-coagulants, inflammatory cytokines, and neutrophil-extracellular traps (NETosis). Within the heart, SARS-CoV-2 infection can directly and indirectly (via cytokine storm) lead to myocardial ischaemia, myocardial infarction, endocardial dysfunction (via inflammation and subsequent fibrosis), and blood stasis in the left atrial atrium (LA) and left atrial appendage (LAA). These can, in turn, lead to intracardiac thrombus. Moreover, thromboinflammation within the vascular beds can drive myocardial injury and vice versa. In the second stage, the dislodgement of thrombus creates mobile embolus, which can be carried to the brain (causing stroke), pulmonary vasculature (causing pulmonary thromboembolism [TE]), or systemically (causing venous thrombosis). Importantly, the presence of thromboembolic complications can lead to progressive COVID-19 disease (in the third conceptual stage). The presence of underlying cardiovascular disease (CVD; i.e., TE) could predispose individuals to SARS-CoV-2 infection via inflammatory derangement. Coexistence of SARS-CoV-2 infection and TE can lead to dysregulated inflammation and coagulation disorders, manifesting with high symptom burden and hospitalization, and increased de novo incidence of TE and other CVDs. Consequently, TE and CVDs predispose COVID-19 patients to worse outcomes, including prolonged intensive care unit (ICU) stay and in-hospital mortality. pericytes, via the ACE2 receptor, are likely to be causative factors, as well as the deleterious effects of the supraphysiological increase of pro-inflammatory factors, the so called “cytokine storm.” of the disease, which likely results in elevated ACS/AMI and thromboembolic risk in COVID-19 patients. Direct viral infection of the endothelium, as well as the surrounding Frontiers in Cardiovascular Medicine | www.frontiersin.org 9 October 2020 | Volume 7 | Article 598400 Roberts et al. Vascular Manifestations of COVID-19 Clinicians and research scientists should consider monitoring the vascular effects of the disease to help identify and manage patients, which may highlight individuals at risk of cardiovascular complications. Despite therapeutic anticoagulation, COVID-19 patients remain at a high risk of both systemic and pulmonary venous thromboembolism. This highlights the need for, perhaps, a more aggressive anticoagulant therapy, and monitoring. Studies should explore the benefits of using D-dimer levels to guide treatment of thromboembolic complications. Further work is needed to determine how best to manage vascular inflammation in COVID-19 patients, which has the potential to significantly improve clinical outcomes in this pandemic. AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. REFERENCES 16. Li B, Yang J, Zhao F, Zhi L, Wang X, Liu L, et al. Prevalence and impact of cardiovascular metabolic diseases on COVID-19 in China. Clin Res Cardiol. (2020) 109:531–8. doi: 10.1007/s00392-020-01626-9 17. Richardson S, Hirsch JS, Narasimhan M, Crawford JM, McGinn T, Davidson KW, et al. 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TNFRSF21 participates in Streptococcus agalactiaeinduced inflammatory necrosis through the NLRP3 inflammasome pathway Lu Yuan Anhui Normal University Jian Ruan Anhui Normal University Mangling Zhang Anhui Normal University Xiaojun Jiang Anhui Normal University Jun Li Anhui Normal University Yafi Cai (  ycai@njau.edu.cn ) Nanjing Agricultural University https://orcid.org/0000-0001-9057-7636 Research Article Keywords: Streptococcus agalactiae, TNFRSF21, Pyroptosis, NLRP3 inflammasome Posted Date: February 23rd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-209964/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/16 Abstract Bovine mastitis caused by Streptococcus agalactiae (S. agalactiae) has resulted in substantial economic loss to the animal husbandry industry; however, the associated molecular mechanism remains unclear and the role of TNFRSF21 in the inflammatory response caused by S. agalactiae is unknown. In this study, we focused on bacterial isolation and identification, histological observations, and provide the first report of the association between TNFRSF21 and pyroptosis. The results showed that S. agalactiaeinduced inflammation can cause increasing damage of the tissue and pyroptosis ratio, significantly increased the level of inflammatory cytokine production (e.g., NLRP3, caspase-1, and IL-1β), and upregulate TNFRSF21 following S. agalactiae infection in a time-dependent manner. In conclusion, these findings indicated that it may fight against the inflammatory response induced by S. agalactiae by inhibiting TNFRSF21 expression and reducing NLRP3 inflammasome pathway-mediated inflammatory necrosis. Introduction Bovine mastitis is caused by inflammation of the udder tissue due to a microbial infection or physical trauma, which is responsible for substantial economic loss to the dairy industry [1,2]. Various pathogenic factors can induce mastitis, including the pasture environment, milking methods, bacteria, and host genotype [3-5]; however, an infection with pathogenic bacteria represents the main cause of mastitis. When pathogenic bacteria invade the mammary gland, the increased permeability of the blood-milk barrier gives rise to a high number of somatic cells in the milk [6]. At the same time, mammary epithelial cells and immune cells work together through pattern recognition receptors that detect pathogen-related molecular patterns to induce a rapid and powerful immune response, which may cause inflammation while removing pathogenic microorganisms [7,8]. At present, S. agalactiae has attracted increased attention due to its high prevalence, and associated risk of inter-species transmission [9], which can lead to a variety of diseases (e.g., dairy cow mastitis, neonatal meningitis, sepsis, and toxic shock) [10,11]. However, the mechanism of S. agalactiae-induced inflammation remains poorly understand. Thus, this study used S. agalactiae to construct inflammatory models to elucidate the associated mechanism. Tumor necrosis factor (TNF) receptors play an important role in the cellular resistance to pathogens and tumor-related immune response, and are key factors of both the inflammatory and immune regulatory responses [12,14]. As a member of the TNF receptor superfamily, TNFRSF21 is highly expressed in a variety of tumor tissues (e.g., ovarian cancer, breast cancer, and adult sarcoma) with potential anticancer effects and can promote the apoptosis of both untransformed and tumor-derived cells [13-15]. In addition, TNFRSF21 has been found to regulate the proliferation and differentiation of immune cells [16,17]. TNFRSF21 has also been shown to have a certain promotional effect on endothelial cell necrosis induced by lupus susceptible mice and autoimmunity caused by tumor cells [18,19]. The activation of nuclear factor-κB (NF-κB) can induce TNFRSF21 expression, the overexpression of which will lead to activation of the NF-κB pathway [15]. The signal mediated by NF-κB is necessary to maintain TNFRSF21 expression in Jurkat leukemia cells [20]. When TNFRSF21 is blocked, the NF-κB signaling pathway is also Page 2/16 inhibited [21]. In contrast, the up-regulation of TNFRSF21 expression can induce HeLa apoptosis [22]. This suggests that TNFRSF21 may mediate the inflammatory responses in tumor progression via NF-κB [23,24]. Coincidentally, once NF-κB is activated, it can regulate the expression of inflammatory genes and cytokine release (e.g., TNF-α, IL-1β, and pro-caspase-1), which assemble on the scattered Golgi apparatus to form NLRP3 inflammasomes, leading to cell pyroptosis and lysis [25-28]. Under normal conditions, pyroptosis represents an important component of the immune response to resist various diseases [29]; however, excessive pyroptosis will inevitably lead to the occurrence of systemic sepsis [30]. Our previous research found that TNSFRSF21 genetic polymorphisms are significantly related to the susceptibility and tolerance of dairy cow mastitis through a genome-wide association analysis [31]. However, the role of TNFRSF21 in the occurrence and development of mastitis remains unknown. In this study, a murine mastitis model of S. agalactiae infection was constructed and we found that NLRP3 was significantly increased and accompanied by upregulated TNFRSF21 expression, indicating that NLRP3 may promote TNFRSF21 expression during the inflammatory response of the breast tissue and jointly promoted pyroptosis. Thus, TNFRSF21 may represent a novel drug target for regulating cellular pyroptosis and relieving inflammation. Materials And Methods Milk sample collection A total of 20 milk samples, including eight normal milk samples and 12 mastitis milk samples, which were collected from the Weigang Animal Diary Company, Jiangsu, China. Bacterial strains S. agalactiae was isolated from fresh milk samples collected from cows with mastitis and injected into the mammary gland tissue of mice to construct a mastitis model. A single colony was isolated from the fresh milk samples using the plate-drawing method and expanded culture to extract the bacterial DNA. After sequencing, multiple pathogenic bacteria (e.g., S. agalactiae) were obtained (primer sequence: F5'AGTTTGATCCTGGCTCAG-3' and R5'-AGGCCCGGGGAACGTATTCAC-3', Sangon Biotech, Shanghai, China). S. agalactiae was incubated on a shaker for 12 h (37°C at 220 rpm), diluted with PBS, and coated on the plate to achieve the desired concentration (1 × 109 CFU/mL), in accordance with previously published methods [27]. The bacteria was stored for a short time at 4°C. Before use, 1 mL of the diluted bacterial solution was resuscitated for 4 h (37°C at 220 rpm), and centrifuged for 5 min (3000 rpm). The supernatant was removed and the bacterial pellet was resuspended in 1 mL PBS. Animals A total of 36 female and 15 male KM mice (6–8-weeks-old, weighing 20 g–25 g) were maintained under SPF housing conditions. Both the female and male mice cohabited at a two female to one male ratio for conception, standard rearing, and free drinking water (25°C ± 1°C, humidity 40%–80%). They were housed Page 3/16 separately after pregnancy until 12 days after calving. The feeding and management of the experimental animals were carried out in accordance with the Animal Management Regulations issued by the National Science and Technology Commission of the People's Republic of China (November 14, 1988), and with the permission of the Animal Ethics Committee of Anhui Normal University (Approval No. 2015002). Mouse model of mastitis Female mice were randomly separated into two groups: 1) S. agalactiae group, one breast was injected with PBS and the other was injected with S. agalactiae after 12 days post-partum; and 2) PBS group, one breast received a blank control and the other was injected with PBS after 12 days post-partum. The pups were removed 2 h prior to the experiment. The female mice were anesthetized with 1% carbrital, and the third and fourth pairs of milk areas were wiped with a cotton ball soaked in 75% alcohol. Slowly, 100 µL of S. agalactiae was injected into the left breast area with a microinjector needle with a diameter of 0.06 mm, and 100 µL of sterile PBS was injected into the right side. At the corresponding time points (24 h, 48 h, and 72 h) after treatment, the mice were sacrificed by cervical dislocation, and the mouse mammary glands were collected under aseptic conditions for subsequent processing and analysis. Histopathological analysis The mammary gland samples of the S. agalactiae group and PBS group were collected and fixed in 4% buffered paraformaldehyde for more than 48 h. the samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E), and the pathological changes were observed by microscopy. Western blot analysis After mixing 400 µL cell lysate and 4 µL proteinase inhibitor, 40 mg of udder tissue were added, ground for 50 s into a powder on an ultrasonic shaker, and lysed for 2.5 h on a shaker. The lysate was centrifuged at 13,000 × g for 20 min and the entire process was carried out at 4°C. Next, the supernatant was collected into a new centrifuge tube and the supernatant was quantified using a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). An appropriate amount of SDS-PAGE loading buffer was added to the protein sample and placed into a metal incubator for 95°C 10 min. The samples were allowed to cool to room temperature and stored at -20°C. After the protein was transferred to the PVDF membrane, it was blocked in 5% BSA for 1 h and incubated with the primary antibody at 4°C overnight. The membranes were then washed three times with TBST for 10 min/wash. The membranes were incubated with a horseradish peroxidase-labeled secondary antibody at 37°C for 1 h. After washing the membrane three times with TBST, an enhanced chemiluminescence solution detection system was used to take pictures and perform an analysis (Tano, Shanghai, China). Densitometry was performed using ImageJ software (National Institutes of Health, Bethesda, MD). The following antibodies were used in this study: TNFRSF21 (Catalog No: #bs-7678R, BOSTER Biotechnology, Wuhan) 68 kDa, NLRP3 (Catalog No: #sc-134306, Santa Cruz Biotechnology, USA) 120 kDa, IL-1β (Catalog No: #sc-12742, Santa Cruz Biotechnology, USA) 17 kDa, caspase1 (Catalog No: #sc-392736, Santa Cruz Biotechnology, USA) 45 kDa, GAPDH (Catalog No: #AT0002, CMCTAG, USA) 36 kDa, goat anti-mouse IgG secondary antibody (Catalog Page 4/16 No: #BA1038, BOSTER Biotechnology, Wuhan), and goat anti-rabbit IgG secondary antibody (Catalog No: #BA1039, BOSTER Biotechnology, Wuhan). Pyroptosis detection Mammary tissue sections (thickness: 4 µm) were deparaffinized and dehydrated by density and fluorescently stained. Densitometry was performed using ImageJ software (National Institutes of Health, Bethesda, MD). The methods were performed in accordance with the manufacturer instructions in the TUNEL cell pyroptosis detection kit-CY3 (50T) (BOSTER, Wuhan, China). Statistical analysis Statistical analyses were performed using a Student’s t-test with GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA, USA). Data were presented as the mean ± the standard deviation (SD). The fluorescence intensity was determined by defining a circular region-of-interest (ROI) for the entire cell. ROIs were corrected based on the average value of a background ROI defined outside the cell. A colocalization analysis was performed using JACoP Plugin in ImageJ. A threshold of P < 0.05 was considered statistically significant. Results Isolation and identification of S. agalactiae S. agalactiae is an epidemic zoonotic pathogen that was initially detected in dairy products. We isolated six strains from 50 single colonies in fresh milk samples by 16S rRNA sequencing, including S. agalactiae, Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Klebsiella pneumoniae. For further confirmation, we cultured Streptococcus in a blood Agar medium exhibiting gray colonies (Fig. 1a). The bacteria were Gram-stained, revealing a purple chain shape (Fig.1b). PCR detection was developed based on -1,4-glusidase gene and the amplified fragment was approximately 1283 bp (Fig.1c). The recovered and purified PCR products were sent for sequencing (Sangon Biotech, Shanghai, China). The maximum similarity of the 16S rRNA sequence between this strain (CSA 001) and S. agalactiae (NR 117503.1) was 99.85%, they were located on the same evolutionary branch, and the Bootstrap value was 100% (Fig.1d). S. agalactiae-induced histopathological impairment of the mammary gland in mice. Mice were sacrificed at 24 h, 48 h, and 72 h after the mammary glands were injected with S. agalactiae. After dissection, the third and fourth pairs of the breast areas were full of milk at 24 h, and no obvious inflammation was observed. At 48 h, the mammary gland areas became swollen and purple. After 72 h, the mammary gland areas on both sides were severely congested, the breast was degenerated, and pus had obviously appeared on the left side (S. agalactiae injection) (Fig. 2). We further fixed the breast tissues, cut them into sections, and stained with H&E. While the acinus in the control group was found to be relatively intact, the acini in the S. agalactiae injection group displayed progressive damage with the Page 5/16 extension of infection time. Furthermore, the injury was more obvious in the breast tissue in the S. agalactiae treatment group compared with the control group. The epithelial cells had sloughed off the acinar structure was gradually destroyed, interstitial thickening was observed, and immune cell infiltration, neutrophils, and exfoliated necrotic breast epithelial cells were scattered in the acinar lumen. Over time, these phenomena became increasingly more serious (Fig.2). S. agalactiae increased the level of IL-1β, NLRP3, caspase-1, and TNFRSF21 expression in the mouse mammary glands. To explore the role of TNFRSF21 in a mouse mastitis model induced by S. agalactiae, we detected the level of TNFRSF21, NLRP3, IL-1β, and caspase-1 protein expression by Western blot. From left to right, Figures 3a and e show the expression for the normal 1, normal 2, control, and treatment groups. Normal 1 and normal 2 were from the same pregnant mouse after stopping breastfeeding for 2 h, with one breast as the blank control and the other injected with PBS in the third pair, which was sacrificed at 24 h, 48 h, and 72 h following PBS injection. The Control and treatment groups were from the same pregnant mouse that had stopping breastfeeding for 2 h, with one breast injected with PBS and the other one injected with S. agalactiae in the third pair, which was sacrificed at 24 h, 48 h, and 72 h after injection, respectively. We found that the level of TNFRSF21, NLRP3, IL-1β, and caspase-1 expression in the normal 1 and normal 2 mice did not change significantly. Compared with the normal group, the level of TNFRSF21 expression in the control group did not change at 24 h, but was higher at 48 h and 72 h (P < 0.01), and showed a significant upward trend from 24 h to 72 h. The level of TNFRSF21 expression was significantly higher in the S. agalactiae treatment group than in the control group at 48 h and 72 h (P < 0.01) (Fig.3b). The level of NLRP3 expression in the treatment group peaked and was extremely significantly different with the other three groups at 24 h (P < 0.001). There was a subsequent downward trend in the level of NLRP3 expression, with an increasingly smaller difference, which was higher compared with the other three groups at any given time (P < 0.01). The level of NLRP3 in the control group peaked at 48 h and were higher than the normal group at 24 h, 48 h, and 72 h (P < 0.05) (Fig.3c). The level of IL-1β expression in the control group increased substantially and was higher than that of the normal 1 and normal 2 groups at 24 h, 48 h, and 72 h (P < 0.05); however, the treatment group expressed the highest level of IL-1β at 48 h, and the difference between the control group gradually became smaller, but remained at significant levels (P < 0.05) (Fig.3d). Compared with the normal 1 and normal 2 groups, the level of caspase-1 expression in the control and treatment groups did not change significantly at 24 h and peaked at 48 h (P < 0.01). Although the level of caspase-1 expression in the control and treatment groups decreased at 72 h, it remained at a higher level than that of the normal group (P < 0.05) (Fig.3f). S. agalactiae triggers pyroptosis in mouse mammary epithelial cells. To further explain whether the inflammatory response of the breast epithelial cells caused by S. agalactiae promotes TNFRSF21-mediated cell pyroptosis, we examined pyroptosis by TUNEL staining. (Fig. 4a, the arrows indicate pyroptosis). These results were confirmed by western blot. With an increase in infection time, the negative control group exhibited potential mammary epithelial cell pyroptosis. The Page 6/16 mammary gland cellular pyroptosis ratios in the S. agalactiae treatment group gradually increased, and were significantly higher than that of the control group (P < 0.001; Fig. 4b). Discussion Due to the diversity and complexity of its pathogenic factors, mastitis has been plaguing the development of global animal husbandry for several years, which is threating to human health. Recently, it has been discovered that S. agalactiae can frequently cause mastitis due to its highly infectious and pathogenic characteristics, [10], clinical neonatal infections, meningitis, endocarditis, and bacteremia [32]. Regarding the pathogenic molecular mechanism of mastitis caused by S. agalactiae, the experts’ views are highly variable, and no conclusions have been reached. Our previous research found that TNSFRSF21 genetic polymorphisms are significantly related to the susceptibility and tolerance of dairy cow mastitis through a genome-wide association analysis [31]; however, the role of TNFRSF21 in S. agalactiae-induced mastitis remains unknown. In this study, we found that the mammary gland tissues of the mice infected with S. agalactiae (bacterial solution with a concentration of 1 × 109 CFU/mL) displayed more severe histopathological damage than that of the control groups, and the level of caspase-1, IL-1β, NLRP3, and TNFRSF21 expression was significantly increased. Interestingly, the control groups also displayed a considerable degree of inflammation. NLRP3 inflammasomes can be detected in many inflammatory diseases, which leads to caspase-1 and IL-1β production [33]. Our results are consistent with studies that have shown that an S. aureus infection can trigger NLRP3 production [34]. As a Gram-positive bacterium, S. agalactiae can also induce high levels of NLRP3 expression, which is accompanied by a surge of IL-1β. These cellular inflammatory factors can cause damage to the breast tissue [35]. Therefore, hematoxylin-eosin staining (H&E) revealed that the mammary tissues of mice infected with S. agalactiae were more severely damaged than that of the control groups. However, the control groups also showed relatively severe damage and up-regulation of inflammatory factors, which may be caused by systemic infection. When there is an imbalance between the normal pyroptosis ratio and recovery ratio, it may lead to decreased barrier integrity, inducing bacterial translocation and the development of systemic sepsis [30]. TNFRSF21 up-regulation, T cell failure, and pyroptosis signals can be observed in patients with viral hepatitis [36]. Although the expression of pyroptosis signals is crucial for the removal of pathogens, an excessive stress response will cause continuous damage [37]. Our results showed that NLRP3, caspase-1, and IL-1β expression was significantly up-regulated during the early stages of infection. However, H&E staining revealed that there was no obvious damage in mouse mammary tissues and PBS group infected by S. agalactiae at 24 h. Furthermore, TNFRSF21 was not significantly up-regulated at 24 h following infection, which may represent the initial stage of the infection when the cell can still repair itself. After 48 h of infection, TNFRSF21 expression in both the control group and S. agalactiae treatment group showed a significant trend towards up-regulation, and the up-regulation of TNFRSF21 induced by S. agalactiae in the breast tissue was more obvious compared with the normal group. At the same time, the fluorescent pyroptosis staining results were also consistent with these findings. Both the control group and the S. Page 7/16 agalactiae stimulation group had no obvious pyroptosis at 24 h; however, the pyroptosis ratio was significantly increased after 48 h. It has been revealed that the cells may enter a developmental phase of pyroptosis during the later stages of infection. It is during these later stages when the immune system is unable to repair the cells that have been over-damaged by the inflammatory response. TNFRSF21 expression was significantly up-regulated, which promoted cellular pyroptosis and tissue necrosis together with NLRP3. Conclusion In this study, S. agalactiae isolated from fresh milk samples was used to establish a mouse model of mastitis in this study. The findings of this study emphasized that S. agalactiae induced pyroptosis in mammary epithelial cells in vivo, and further promoted TNFRSF21 up-regulation through activation of the NLRP3 inflammasome and IL-1β expression, which aggravates tissue damage. However, there was no evidence that TNFRSF21 was a direct factor that led to pyroptosis in the later stages of the NLRP3 inflammasome. Further understanding of the role of S. agalactiae in the pathogenesis of mastitis will provide clues for the development of new treatment strategies to prevent disease. Declarations Funding This experiment was supported by The Agricultural Innovation fund of Jiangsu Province [grant no. CX (18)3014]; National Key R&D Program of China (grant No. 2018YFC1200201); National Natural Science Foundation of China (grant No. 31970413); Anhui Natural Science Foundation (grant No. 1908085MC83); and Start-up grant from Nanjing Agricultural University (grant No. 804090). Conflict of interest All authors declare that they have no conflict of interest. Availability of data and material The datasets used or analysed during the current study are available from the corresponding author on reasonable request. Ethics approval The experimental protocol was established, according to the ethical guidelines of the Declaration and was approved by the Ethics Committee of the Animal Management Regulations issued by the National Science and Technology Commission of the People's Republic of China (November 14, 1988), and with the permission of the Animal Ethics Committee of Anhui Normal University (Approval No. 2015002). Consent to participate Not applicable. Consent for publication Not applicable. References Page 8/16 1. Heikkila, A.M., J.I. Nousiainen, and S. Pyorala, Costs of clinical mastitis with special reference to premature culling. J Dairy Sci, 2012. 95(1): p. 139-50. 2. Deb, R., et al., Trends in diagnosis and control of bovine mastitis: a review. Pak J Biol Sci, 2013. 16(23): p. 1653-61. 3. De Vliegher, S., et al., Invited review: Mastitis in dairy heifers: nature of the disease, potential impact, prevention, and control. J Dairy Sci, 2012. 95(3): p. 1025-40. 4. Klaas, I.C. and R.N. Zadoks, An update on environmental mastitis: Challenging perceptions. Transbound Emerg Dis, 2018. 65 Suppl 1: p. 166-185. 5. Gunther, J., et al., Comparison of the pathogen species-specific immune response in udder derived cell types and their models. Vet Res, 2016. 47: p. 22. 6. Wellnitz, O., et al., Short communication: Differential loss of bovine mammary epithelial barrier integrity in response to lipopolysaccharide and lipoteichoic acid. J Dairy Sci, 2016. 99(6): p. 48514856. 7. Takeda, K. and S. Akira, Toll-like receptors in innate immunity. Int Immunol, 2005. 17(1): p. 1-14. 8. Mifsud, E.J., A.C. Tan, and D.C. Jackson, TLR agonists as modulators of the innate immune response and their potential as agents against infectious Disease. Front Immunol, 2014. 5: p. 79. 9. Foxman, B., et al., Risk factors for group B streptococcal colonization: potential for different transmission systems by capsular type. Ann Epidemiol, 2007. 17(11): p. 854-62. 10. Pang, M., et al., Molecular and virulence characterization of highly prevalent Streptococcus agalactiae circulated in bovine dairy herds. Vet Res, 2017. 48(1): p. 65. 11. Sunkara, B., et al., Group B Streptococcus infections in non-pregnant adults: the role of immunosuppression. Int J Infect Dis, 2012. 16(3): p. e182-6. 12. Ward-Kavanagh, L.K., et al., The TNF receptor superfamily in co-stimulating and co-inhibitory responses. Immunity, 2016. 44(5): p. 1005-19. 13. Stegmann, S., et al., Death receptor 6 (DR6) is overexpressed in astrocytomas. Anticancer Res, 2019. 39(5): p. 2299-2306. 14. Pan, G., et al., Identification and functional characterization of DR6, a novel death domain-containing TNF receptor. FEBS Lett, 1998. 431(3): p. 351-6. 15. Kasof, G.M., et al., Tumor necrosis factor-alpha induces the expression of DR6, a member of the TNF receptor family, through activation of NF-kappaB. Oncogene, 2001. 20(55): p. 7965-75. 16. DeRosa, D.C., et al., Tumor-derived death receptor 6 modulates dendritic cell development. Cancer Immunol Immunother, 2008. 57(6): p. 777-87. 17. Schmidt, C.S., et al., Enhanced B cell expansion, survival, and humoral responses by targeting death receptor 6. J Exp Med, 2003. 197(1): p. 51-62. 18. Strilic, B., et al., Tumour-cell-induced endothelial cell necroptosis via death receptor 6 promotes metastasis. Nature, 2016. 536(7615): p. 215-8. Page 9/16 19. Fujikura, D., et al., Death receptor 6 contributes to autoimmunity in lupus-prone mice. Nat Commun, 2017. 8: p. 13957. 20. Klima, M., et al., T-cell activation triggers death receptor-6 expression in a NF-kappaB and NF-AT dependent manner. Mol Immunol, 2011. 48(12-13): p. 1439-47. 21. Zhang, D., et al., MiR-210 inhibits NF-kappaB signaling pathway by targeting DR6 in osteoarthritis. Sci Rep, 2015. 5: p. 12775. 22. Dong, Y., et al., Lysophosphatidic acid triggers pyroptosis in HeLa cells through the upregulation of tumor necrosis factor receptor superfamily member 21. Mediators Inflamm, 2017. 2017: p. 2754756. 23. Yang, X., et al., Death receptor 6 (DR6) is required for mouse B16 tumor angiogenesis via the NFkappaB, P38 MAPK and STAT3 pathways. Oncogenesis, 2016. 5: p. e206. 24. Cui, L., Y. Han, and Z. Dong, Original paper miR-106a mimics the nuclear factor-κB signalling pathway by targeting DR6 in rats with osteoarthritis. Archives of Medical Science, 2020. 25. Wang, J.E., et al., Peptidoglycan and lipoteichoic acid from Staphylococcus aureus induce tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-10 production in both T cells and monocytes in a human whole blood model. Infect Immun, 2000. 68(7): p. 3965-70. 26. Bi, C.L., et al., Selenium inhibits Staphylococcus aureus-induced inflammation by suppressing the activation of the NF-kappaB and MAPK signalling pathways in RAW264.7 macrophages. Eur J Pharmacol, 2016. 780: p. 159-65. 27. Breyne, K., et al., Efficacy and safety of a bovine-associated Staphylococcus aureus phage cocktail in a murine model of mastitis. Front Microbiol, 2017. 8: p. 2348. 28. Chen, J. and Z.J. Chen, PtdIns4P on dispersed trans-Golgi network mediates NLRP3 inflammasome activation. Nature, 2018. 564(7734): p. 71-76. 29. Peterson, L.W., et al., RIPK1-dependent pyroptosis bypasses pathogen blockade of innate signaling to promote immune defense. J Exp Med, 2017. 214(11): p. 3171-3182. 30. Hunter, C.J., et al., Enterobacter sakazakii enhances epithelial cell injury by inducing pyroptosis in a rat model of necrotizing enterocolitis. J Infect Dis, 2008. 198(4): p. 586-93. 31. Yang, F., et al., Three novel players: PTK2B, SYK, and TNFRSF21 were identified to be involved in the regulation of bovine mastitis susceptibility via GWAS and post-transcriptional analysis. Front Immunol, 2019. 10: p. 1579. 32. Lyhs, U., et al., Streptococcus agalactiae Serotype IV in humans and cattle, Northern Europe(1). Emerg Infect Dis, 2016. 22(12): p. 2097-2103. 33. Mangan, M.S.J., et al., Targeting the NLRP3 inflammasome in inflammatory diseases. Nat Rev Drug Discov, 2018. 17(9): p. 688. 34. Zhao, G., et al., Polydatin reduces Staphylococcus aureus lipoteichoic acid-induced injury by attenuating reactive oxygen species generation and TLR2-NFkappaB signalling. J Cell Mol Med, 2017. 21(11): p. 2796-2808. Page 10/16 35. Zhao, X. and P. Lacasse, Mammary tissue damage during bovine mastitis: causes and control. J Anim Sci, 2008. 86(13 Suppl): p. 57-65. 36. Barathan, M., et al., Chronic hepatitis C virus infection triggers spontaneous differential expression of biosignatures associated with T cell exhaustion and pyroptosis signaling in peripheral blood mononucleocytes. Pyroptosis, 2015. 20(4): p. 466-80. 37. Sadek, K., E. Saleh, and M. Ayoub, Selective, reliable blood and milk bio-markers for diagnosing clinical and subclinical bovine mastitis. Trop Anim Health Prod, 2017. 49(2): p. 431-437. Figures Page 11/16 Figure 1 Isolation and identification of S. agalactiae. Identification of S. agalactiae: a The grayish-white colonies were grown in Colombian blood Agar culture medium. b Gram staining revealed purple chain bacteria. c A PCR assay was conducted based on the α-1,4-glucosidase gene, which was positive (1283 bp). d Sequencing: bacteria CSA 001 (1283 bp) and S. agalactiae (NR 117503.1) were the same bacteria, with a confidence level of 100%. Page 12/16 Figure 2 S. agalactiae-induced histopathological impairment in mice. S. agalactiae was injected into the third and fourth pair of mammary glands on the left, and PBS was injected into the third and fourth pair of mammary glands on the right. H&E staining of breast histology: control group and treatment group (injected with S. agalactiae). Page 13/16 Figure 3 S. agalactiae increased the level of IL-1 β, NLRP3, caspase-1, and TNFRSF21 expression in mice. The mammary gland tissues of each group (n ≥ 3) were collected at 24 h, 48 h, and 72 h after S. agalactiae and PBS infection. a and e The level of IL-1 β, NLRP3, caspase-1, and TNFRSF21 protein expression in the mice was detected by Western blot. b, c, d, and f The relative IL-1 β, NLRP3, caspase-1, and TNFRSF21 protein levels were quantified using Image J software and normalized to GAPDH expression in mice. Data are presented as the means ± the standard deviation (SD) (n ≥ 3) (*P < 0.05; **P < 0.01; ***P < 0.001). Page 14/16 Figure 4 S. agalactiae induced pyroptosis in the mammary epithelial cells of the mice. a TUNEL staining of pyroptosis in the mouse mammary gland tissues in each group (n ≥ 3). b The percentage of pyroptosis in the mouse breast tissue. Data are presented as the means ± the standard deviation (SD) (n ≥ 3) (***P < 0.001). Page 15/16 Figure 5 S. agalactiae-stimulated mammalian mammary gland epithelium-induced pyroptosis via activation of the NLRP3 pathway and excessive TNFRSF21 expression. S. agalactiae was isolated from bovine mastitis milk samples, which was used to infect the mice. The S. agalactiae-infected mammary gland epithelium activated the NLRP3 inflammasome and induced excessive TNFRSF21 expression; however, there was no evidence to support the relationship between NLRP3 inflammasome activation and TNFRSF21. Finally, pyroptosis was observed in the mammary epithelial cells. Page 16/16 

W3099209921.txt

Published for SISSA by Springer Received: March 5, Revised: May 7, Accepted: May 23, Published: May 31, 2018 2018 2018 2018 Seungwon Baek,a,b Arindam Dasa and Takaaki Nomuraa a School of Physics, KIAS, Seoul 02455, Korea b Department of Physics, Korea University, Seoul 02841, Korea E-mail: sbaek@korea.ac.kr, arindam@kias.re.kr, nomura@kias.re.kr Abstract: We consider a neutrino Two Higgs Doublet Model (νTHDM) in which neutrinos obtain naturally small Dirac masses from the soft symmetry breaking of a global U(1)X symmetry. We extended the model so the soft term is generated by the spontaneous breaking of U(1)X by a new scalar field. The symmetry breaking pattern can also stabilize a scalar dark matter candidate. After constructing the model, we study the phenomenology of the dark matter: relic density, direct and indirect detection. Keywords: Beyond Standard Model, Higgs Physics, Neutrino Physics ArXiv ePrint: 1802.08615 Open Access, c The Authors. Article funded by SCOAP3 . https://doi.org/10.1007/JHEP05(2018)205 JHEP05(2018)205 Scalar dark matter search from the extended νTHDM Contents 1 Introduction 1 2 The Model 2 3 DM phenomenology 6 1 12 Introduction The existence of the tiny neutrino mass can be naturally explained by the seesaw mechanism [1–7] which extends the Standard Model (SM) through Majorana type Right Handed Neutrinos (RHNs). As a result the SM light neutrinos become Majorana particles. Alternatively there is a simple model, neutrino Two Higgs Doublet Model (νTHDM) [8, 9], which can generate the Dirac mass term for the light neutrinos as well as for the other fermions in the SM. In this model we have two Higgs doublets; one is the same as the SM-like Higgs doublet and the other one is having a small VEV (O(1)) eV to explain the tiny neutrino mass correctly. Due to this fact, the neutrino Dirac Yukawa coupling could be order 1. It has been discussed in [8] that a global softly broken U(1)X symmetry can forbid the Majorana mass terms of the RHNs; a hidden U(1) gauge symmetry can be also applied to realize νTHDM as in ref. [10]. In this model all the SM fermions obtain Dirac mass terms via Yukawa interactions with the SM-like Higgs doublet (Φ2 ) whereas only the neutrinos get Dirac masses through the Yukawa coupling with the other Higgs doublet (Φ1 ). Another scenario of the generation of Dirac neutrino mass through a dimension five operator has been studied in [11]. The corresponding Yukawa interactions of the Lagrangian can be written as e 2 u R − Q L Y d Φ2 d R − L L Y e Φ2 e R − L L Y ν Φ e 1 νR + H.c. LY = −QL Y u Φ (1.1) e i = iσ2 Φ∗ (i = 1, 2), QL is the SM quark doublet, LL is the SM lepton doublet, where Φ i eR is the right handed charged lepton, uR is the right handed up-quark, dR is the right handed down-quark and νR are the RHNs. The Φ1 and νR are assigned with the global charge 3 under the U(1)X group. The global symmetry forbids the Majorana mass term between the RHNs. In the original model [8], the global symmetry is softly broken by the mixed mass term between Φ1 and Φ2 (m212 Φ†1 Φ2 ) such that a small VEV is obtained by seesaw-like formulas m2 v2 v1 = 122 , (1.2) MA –1– JHEP05(2018)205 4 Conclusion Scalar Fields Φ2 S 2 1 X 1 New Fermion νR 1 SU(2)L Φ1 2 U(1)Y 1 2 1 2 0 0 0 U(1)X 3 0 3 1 3 Table 1. Scalar fields and new fermion in our model. 2 The Model We discuss the extended version of the model in [8] with a scalar field (X). We write the scalar and the RHN sectors of the particle content in table 1 The gauge singlet Yukawa interaction between the lepton doublet (LL ), the doublet scalars (Φ1 , Φ2 ) and the RHNs (νR ) can be written as L ⊃ −Yije L̄Li Φ2 eRj − Yijν L̄Li Φ̃1 νRj + H.c. (2.1) We assume that the Yukawa coupling constants Yije and Yijν are real. The scalar potential can be written by 2 V (Φ1 , Φ2 , S) = −m211 Φ†1 Φ1 − m222 Φ†2 Φ2 − m2S S † S + MX X † X − (µΦ†1 Φ2 S + h.c.) + λ1 (Φ†1 Φ1 )2 + λ2 (Φ†2 Φ2 )2 + λ3 (Φ†1 Φ1 )(Φ†2 Φ2 ) + λ4 (Φ†1 Φ2 )(Φ†2 Φ1 ) + λS (S † S)2 + λ1S Φ†1 Φ1 S † S + λ2S Φ†2 Φ2 S † S + λX (X † X)2 + λ1X Φ†1 Φ1 X † X + λ2X Φ†2 Φ2 X † X + λSX S † SX † X − (λ3X S † XXX + H.c.). (2.2) The Dirac mass terms of the neutrinos are generated by the small VEV of Φ1 . According to [8, 9] we assume that the VEV of Φ1 is much smaller than the electroweak scale. The –2– JHEP05(2018)205 where MA is the pseudo-scalar mass in [8]. If MA ∼ 100 GeV and m12 ∼ O(100) keV then v1 can be obtained as O(1) eV. In the paper [12], the model is extended to include singlet scalar S which breaks the U(1)X symmetry. The soft term m212 is identified with µhSi where µ is the Higgs mixing term, µΦ†1 Φ2 S + h.c.. It has been studied in [12] that an SM singlet fermion being charged under U(1)X could be a potential DM candidate. In this paper we extend the model with a natural scalar Dark Matter (DM) candidate (X). In this model the global U(1)X symmetry is spontaneously broken down to Z2 symmetry by VEV of a new singlet scalar S. The remnant of the Z2 symmetry makes the DM candidate stable. The Z2 symmetry would be broken by quantum gravity effect and DM would decay via effective interaction [13]. This can be avoided if the U(1)X is a remnant of local symmetry at a high energy scale and we assume the Z2 symmetry is not broken. A CP odd component of S becomes the Goldstone boson and hence we study the DM annihilation from this model and compare with the current experimental sensitivity. The papers is organized as follows. In section 2 we describe the model. In section 3 we discuss the DM phenomenology and finally in section 4 we conclude. X = X 0e a i 2vS S , Φ1 = Φ01 e a i vS S , a 1 i S S = √ rS e vS , 2 (2.4) where rS = ρ + vS . We assume X does not develop a VEV while the VEVs of Φ1 , Φ2 and S are obtained by requiring the stationary conditions ∂V (v1 , v2 , vS )/∂vi = 0 following √ −2m211 v1 + 2λ1 v13 + v1 (λ1S vS2 + λ3 v22 + λ4 v22 ) − 2µv2 vS = 0, √ −2m222 v2 + 2λ2 v23 + v2 (λ2S vS2 + λ3 v12 + λ4 v12 ) − 2µv1 vS = 0, √ −2m2S vS + 2λS vS3 + vS (λ1S v12 + λ2S v22 ) − 2µv1 v2 = 0. (2.5) We then find that these conditions can be satisfied with v1 ' µ  {v2 , vS } and SM Higgs VEV is given as v ' v2 ' 246 GeV. From the first one of the eq. (2.5) we find that v1 is proportional to and of the same order with µ such that √ 2µv2 vS v1 ' . (2.6) 2 λ1S vS + (λ3 + λ4 )v22 − 2m211 The small order of v1 (∼ µ) is required to keep v2 and vS in the electroweak scale. Considering the neutrino mass scale as mν ∼ 0.1 eV, the value of µ/v2 should be small such as µ/v2 ∼ O(10−12 ) ensuring Y ν as O(1) such that me /v2 ∼ O(10−6 ). Hence v1 is considered to be smaller than the other VEVs. It also interesting to notice that µ = 0 restores the symmetry of the Lagrangian hence a technically natural small value of µ is acceptable [15, 16]. It is also interesting to notice that µ = 0 enhances the symmetry of the Lagrangian in the sense that we can assign arbitrary U(1)X charge to Φ1 , which ensures the radiative generation of the µ-term is proportional to µ itself. Hence a small value of µ is technically natural [15, 16]. Now we identify mass spectra in the scalar sector. ± Charged scalar: in this case we calculate the mass matrix in the basis (φ± 1 , φ2 ) where ± φ± 1 is approximately physical charged scalar while φ2 is approximately NG boson absorbed by W ± boson. In the following we write physical charged scalar field as H ± ' φ± 1 . The charged scalar mass matrix can be written as √ ! ! √ √ v2 ( 2µvS −λ4 v1 v2 ) 1 v2 ( 2µvS −λ4 v1 v2 ) − ( 2µv − λ v v ) 0 4 1 2 S 2 2 √ 2v1 MH ' . (2.7) √ 2v1 ± = v1 ( 2µvS −λ4 v1 v2 ) 0 0 − 12 ( 2µvS − λ4 v1 v2 ) 2v 2 The charged Higgs mass can be written as √ v2 ( 2µvS − λ4 v1 v2 ) 2 mH ± ' . 2v1 –3– (2.8) JHEP05(2018)205 vacuum stability analysis of a general scalar potential has been studied in [14]. Additionally, a remaining Z3 symmetry is also involved when U(1)X is broken by non-zero VEV of S. Here X is the only Z3 charged stable (scalar) particle and as a result X could be considered as a potential Dark Matter (DM) candidate. The mass term MX of X in eq. (2.2) is positive definite which forbids X to get VEV and as a result the Z3 symmetry promotes the stability of X as a DM candidate. It has already been discussed in [12] that a CP-odd component in S becomes massless Goldstone boson. Then we write scalar fields as follows ! ! φ+ φ+ 1 2 0 Φ1 = , Φ2 = , (2.3) √1 (v1 + h1 + ia1 ) √1 (v2 + h2 + ia2 ) 2 2 We find that all the masses of the mass eigenstates, Hi (i = 1, 2, 3), are at the electroweak scale and the mixings between h1 and other components are negligibly small while the h2 and ρ can have sizable mixing. The mass eigenvalues and the mixing angle for h2 and ρ system can be given by   q 1 2 2 2 2 2 4 2 mH2 ,H3 = m22 + m33 ∓ (m22 − m33 ) + 4m23 , (2.10) 2 −2m223 tan 2θ = 2 , (2.11) m22 − m233 m222 = 2λ2 v22 , m233 = 2λS vS2 , m223 = λ2S v2 vS . Hence the mass eigenstates are obtained as      H1 1 0 0 h1      H2  ' 0 cos θ − sin θ h2  . H3 0 sin θ cos θ ρ (2.12) (2.13) Here H2 is the SM-like Higgs, h, and mH2 ' mh where the mixing angle θ between H2 and H3 is constrained as sin θ ≤ 0.2 by the LHC Higgs data [17–19] using the numerical analyses on the Higgs decay followed by [20, 21]. CP-odd neutral scalar: calculating the mass matrix of the pseudo-scalars in a basis (a1 , a2 , aS ) we get the mass matrix as v v   µv v  2 S √2 S 0 0 −v −v 2 S µ  v1   2v1  (2.14) MA2 = √ −vS v1vv2S v1  '  0 0 0 , 2 −v2 v1 vv1Sv2 0 00 S √ using S ' vS +ρ+ia . In the last step we used the approximation, v1 (∼ µ)  v2 , vS . We 2 find three mass eigenstates, A = a1 − v1 v1 a2 − aS , v2 vS v1 a1 + a2 , v2   v1 v12 v12 a= a1 − a2 + 1 + 2 aS , vS v2 vS v2 G0 = –4– (2.15) JHEP05(2018)205 CP-even neutral scalar: in the case of CP-even scalar all three components are physical. Hence the mass matrix can be written in the basis of (h1 , h2 , ρ) as   √ 2 vS √ S λ1S v1 vS − µv √2 2λ1 v12 + µv (λ3 + λ4 )v1 v2 − µv 2v1 2 2  µv 2 √ 1 vS √1  2λ2 v22 + µv λ2S v2 vS − µv MH = (λ3 + λ4 )v1 v2 − √2S 2v2 2 µv µv µv v 2 2 1 1 λ1S v1 vS − √2 λ2S v2 vS − √2 2λS vS + √2v 2 S  µv v  √2 S 0 0   2v1 (2.9) ' 0 2λ2 v22 λ2S v2 vS  . 2 0 λ2S v2 vS 2λS vS up to normalization. They correspond to massive pseudo-scalar, the masslesss NambuGoldstone (NG) mode which is absorbed by the Z boson, and a massless physical Goldstone boson associated with the U(1)X breaking, respectively. Hence the mass of A is given by m2A = µ(v12 v22 + v12 vS2 + v22 vS2 ) µv2 vS √ ' √ , 2v1 v2 vS 2v1 (2.16) − λ2S mf ∂µ a∂ µ af¯f, 2m2H3 m2H2 (2.17) where mf is the mass of the SM fermion f , and we used as ' a. The temperature, Ta , at which a decouples from thermal bath is roughly estimated by [22] λ22S m2f Ta5 mP L collision rate ' ∼ 1, expansion rate m4H2 m4H3 (2.18) where mP L denotes the Planck mass and mf should be smaller than Ta so that f is in thermal bath. The decoupling temperature is then calculated by  m  4  GeV  25  0.01  25 5 H3 Ta ∼ 2 GeV . mf λ2S 100 GeV (2.19) Thus Goldstone boson a can decouple from thermal bath sufficiently earlier than muon decoupling and does not contribute to the effective number of active neutrinos 1 [23]. Note that the Goldstone boson should be in thermal bath at temperature below that of freeze-out of DM when we consider the relic density of DM, X, is explained by the process, X X̄ → aa, in our analysis below. Taking minimum DM mass as ∼ 100 GeV freeze-out temperature Tf is larger than ∼ 100/xf GeV ∼ 4 GeV where xf = mDM /Tf ∼ 25. Therefore we can get Tf > Ta even with small λ2S (= 0.01) as long as mH3 is not much heavier than the electroweak scale. As the phenomenology of the Higgs sector has been discussed in [8, 12, 24, 25], we concentrate on the DM phenomenology in the following analysis. 1 If mH3 ≈ 500 MeV and λ2S ≈ 0.005, then a can make sizable contribution: ∆Neff = 4/7 [22]. –5– JHEP05(2018)205 which is at the electroweak scale. It can be shown [12] that the Goldstone boson, a, is safe from the phenomenological constraints such as Z → Hi a(i = 1, 2, 3) decay, stellar cooling from the interaction aeγ5 e, etc., because it interacts with the SM particles only via highlysuppressed (∼ v1 /v2,S ) mixing with the SM Higgs. Note that, in our analysis below, we approximate pseudo-scalars as A ' a1 , G0 ' a2 and a ' aS since we assume v1  v2 , vS in realizing small neutrino mass. Here we also discuss decoupling of the physical Goldstone boson from thermal bath where we assume it is thermalized via Higgs portal interaction. The interactions ρ∂µ aS ∂ µ aS /vS , λ2S vS v2 ρh2 and the SM Yukawa interactions generate the effective interaction among the Goldstone boson a and the SM fermions 3 DM phenomenology In this section, we discuss DM physics of our model such as relic density, direct and indirect detections which are compared with experimental constraints. Since the Higgs portal interaction is strongly constrained by DM direct detection [26–29], we consider the case of small mixing so that h1 ' H1 , h2 ' H2 and ρ ' H3 ; here H2 is the SM-like Higgs in our DM analysis. Dark matter interaction. Firstly masses of dark matter candidates X is given by [27] 2 m2X = MX + λ1X 2 λ2X 2 λSX 2 v + v + v 2 1 2 2 2 S (3.1) where the real and imaginary part of X has the same mass and X is taken as a complex scalar field; this is due to remnant Z3 symmetry. The interactions relevant to DM physics are given by L⊃ 1 1 ∂µ a(X∂ µ X ∗ − X ∗ ∂ µ X) + 2 ∂µ a∂ µ aX ∗ X vS 4vS   λ1X 1 2 λ2X + − 2 + H H + H1 + A X ∗ X + (2v2 H2 + H22 )X ∗ X 2 2 4 λSX λ3X + (2vS H3 + H32 )X ∗ X + (vS + H3 )(XXX + c.c.) 4 2   1 1 µ2S − µSS H33 + H3 ∂µ a∂ µ a − µ1S H3 H + H − + (H12 + A2 ) − H3 H22 , vS 2 2 (3.2) where we ignored terms proportional to v1 since the value of VEV is tiny, µSS ≡ m2H3 /(2vS ), µ1S ≡ λ1S vS , µ2S ≡ λ2S vS , and omitted scalar mixing sin θ(cos θ) assuming cos θ ' 1 and sin θ  1. Thus relevant free parameters to describe DM physics are summarized as; {mX , mH1 , mH3 , mA , mH ± , vS , λ1X , λ2X , λSX , λ3X , µ1S , µ2S }, –6– (3.3) JHEP05(2018)205 Figure 1. Diagrams in (I), (II), (III) and (IV) correspond to DM annihilation process in scenario-I, II, III and IV. Scenario-I 2000 VS @GeVD 1500 1000 100 200 300 mX @GeVD 400 500 Figure 2. Scatter plot for parameters on mX -vS plane under the DM relic abundance bound in Scenario-I. where we choose µ1S,2S as free parameter instead of λ1S,2S and we use µSS = m2H3 /(2vS ). In our analysis, we focus on several specific scenarios for DM physics by making assumptions for model parameters to illustrate some particular processes of DM annihilations. These scenarios are given as follows: • Scenario-I: 100 GeV < vS < 2000 GeV, {λ1X , λ2X , λSX , λ3X , µ1S /v}  1. • Scenario-II: vS  v, {λSX , µ1S /v}  {λ1X , λ2X , λ3X , µ1S /v}. • Scenario-III: vS  v, λ1X  {λ2X , λSX , λ3X , µ1S /v}. • Scenario-IV: vS  v, λX3  {λ1X , λ2X , λSX , µ1S /v}. Here we set v ≡ v2 ' 246 GeV since v1  v2 . In scenario-I DM mainly annihilates into aS aS and aS H3 final state as shown in figure 1-(I). In scenario-II DM annihilates via H3 portal interaction as figure 1-(II). In scenario-III DM annihilates into components of Φ1 through contact interaction with coupling λ1X as shown figure 1-(III). Finally scenarioIV represents semi-annihilation processes XX → XH3 as shown in figure 1-(IV). In our analysis, we assumed λ2S  O(1) so that we can neglect the case of DM annihilation via the SM Higgs portal interaction since it is well known and constraints from direct detection experiments are strong. Relic density. Here we estimate the thermal relic density of DM for each scenario given above. The relic density is calculated numerically with micrOMEGAs 4.3.5 [30] to solve the Boltzmann equation by implementing relevant interactions. In numerical calculations we apply randomly produced parameter sets in the following parameter ranges. For all –7– JHEP05(2018)205 500 Scenario-II Scenario-II 1.000 0.500 1.000 0.100 0.050 0.100 0.500 ΛSX ΛSX 0.050 0.010 0.005 0.005 100 200 300 mX @GeVD 400 0.001 500 200 400 600 Μ1 S @GeVD 800 1000 Figure 3. Scatter plot for parameters on mX -λSX and µ1S -λSX planes in left and right panels under the DM relic abundance bound in Scenario-II. Scenario-IV 1.00 0.50 0.50 0.20 0.20 Λ3 X Λ1 X Scenario-III 1.00 0.10 0.10 0.05 0.05 0.02 0.02 100 200 300 mX @GeVD 400 500 100 200 300 400 500 MX @GeVD Figure 4. Left: scatter plot for parameters on mX -λ1X plane under the DM relic abundance bound in Scenario-III. Right: that for parameters on mX -λ12X in Scenario-IV. scenarios we apply parameter settings as mX ∈ [50, 500] GeV, µ2S = 1 GeV, MH1 = MA = MH ± ∈ [100, 1000] GeV, λ2X  1, (3.4) where the setting for λ2X is to suppress the SM Higgs portal interactions and small value of µ2S is to suppress scalar mixing. Then we set parameter region for each scenarios as follows: Scenraio−I : vS ∈ [100, 2000] GeV, λSX,1X,3X ∈ [10−8 , 10−4 ], µ1S ∈ [0.001, 0.1] GeV, MH3 ∈ [10, 30] GeV, –8– (3.5) JHEP05(2018)205 0.001 0.010 Scenario−II : vS ∈ [3000, 10000] GeV, µ1S ∈ [100, 1000] GeV, Scenario−III : vS ∈ [3000, 10000] GeV, µ1S ∈ [0.001, 0.1] GeV, Scenario−IV : vS ∈ [3000, 10000] GeV, µ1S ∈ [0.001, 0.1] GeV, λSX ∈ [10−3 , 1], λ1X,3X ∈ [10−8 , 10−4 ], MH3 ∈ [150, 2000] GeV, λ1X ∈ [10 −3 , 1], (3.6) λSX,3X ∈ [10 −8 λSX,1X ∈ [10 −8 MH3 ∈ [150, 2000] GeV, λ3X ∈ [10 −3 , 1], , 10−4 ], (3.7) MH3 ∈ [50, mX ] GeV. , 10−4 ], (3.8) 0.11 . Ωh2 . 0.13 . (3.9) In figure 2, we show parameter points on mX -vS plane which can explain the observed relic density of DM in Scenario-I. In this scenario, relic density is mostly determined by the cross section of XX → aS aS process which depends on mX /vS via second term of the Lagrangian in eq. (3.2). Thus preferred value of vS becomes larger when DM mass increases as seen in figure 2. In left and right panel of figure 3, we respectively show parameter points on mX -λSX and µ1S -λSX planes satisfying correct relic density in Scenario-II. In this scenario, the region mX . 100 GeV requires relatively larger λSX coupling since scalar boson modes {H3 H3 , H1 H1 , AA, H ± H ∓ } are forbidden by our assumption for scalar boson masses. On the other hand the region mX > 100 GeV allow wider range of λSX around 0.01 . λSX . 1.0 since DM can annihilate into other scalar bosons if kinematically allowed. In left (right) panel of figure 4, we show parameter region on mX -λ1X (λ3X ) satisfying the relic density in Scenario-III(IV). In scenario-III, DM mass should be larger than ∼ 100 GeV to annihilate into scalar bosons from Φ1 and required value of the coupling is 0.2 . λ1X . 1.0 for mX ≤ 500 GeV. In scenario-IV, the required value of the coupling λ3X has similar behavior as λ1X in the scenario-III for mX > 100 GeV but slightly larger value. This is due to the fact that semi-annihilation process require larger cross section than that of annihilation process. Direct detection. Here we briefly discuss constraints from direct detection experiments estimating DM-nucleon scattering cross section in our model. Then we focus on our scenario-III since DM can have sizable interaction with nucleon via H2 and H3 exchange and investigate upper limit of mixing sin θ. The relevant interaction Lagrangian with mixing effect is given by L⊃ X mq λSX vS ∗ X X(cθ H3 − sθ H2 ) + q̄q(sθ H3 + cθ H2 ), 2 v q (3.10) where q denote the SM quarks with mass mq , and we assumed µX  λSX vS as in the relic density calculation. We thus obtain the following effective Lagrangian for DM-quark interaction by integrating out H2 and H3 ; ! X λSX vS mq sθ cθ 1 1 Leff = X ∗ X q̄q, (3.11) 2 − m2 2v m H3 h q –9– JHEP05(2018)205 Then we search for the parameter sets which can accommodate with observed relic density. Here we apply an approximated region [31] where mH2 ' mh = 125 GeV is used. The effective interaction can be rewritten in terms of nucleon N instead of quarks such that ! fN λSX vS mN sθ cθ 1 1 Leff = − X ∗ X N̄ N, (3.12) v m2h m2H3 where mN is nucleon mass and fN is the effective coupling constant given by X X mq fN = fqN = hN |q̄q|N i. m N q q q=c,b,t (3.14) q=c,b,t which is obtained by calculating the triangle diagram for heavy quarks inside a loop. Then we write the trace of the stress energy tensor as follows by considering the scale anomaly; θµµ = mN N̄ N = X mq q̄q − q 7αs a aµν G G . 8π µν (3.15) Combining eqs. (3.14) and (3.15), we get  X fqN = q=c,b,t  2 1− 9 X fqN  , (3.16) q=u,d,s which leads fN = 2 7 X N + fq . 9 9 (3.17) q=u,d,s Finally we obtain the spin independent X-N scattering cross section as follows; !2 2 m2 λ2 v 2 s2 c2 1 µ2N X fN 1 1 N SX S θ θ σSI (XN → XN ) = − , 8π v 2 m2X m2h m2H3 (3.18) where µN X = mN mX /(mN + mX ) is the reduced mass of nucleon and DM. Here we consider DM-neutron scattering cross section for simplicity where that of DM-proton case gives almost similar result. In this case, we adopt the effective coupling fn ' 0.287 (with fun = 0.0110, fdn = 0.0273, fsb = 0.0447) in estimating the cross section. In figure 5, we show DM-nucleon scattering cross section as a function of sin θ we take mX = 300 GeV, mH3 = 300 GeV, vS = 5000 GeV, and λSX = 0.5(0.01) for red(blue) line as reference values. We find that some parameter region is constrained by direct detection when λSX is relatively large and sin θ > 0.01. More parameter region will be tested in future direct detection experiments. The Higgs portal interaction can be also tested by collider experiments. The interaction can be tested via searches for invisible decay of the SM Higgs for 2mX < mh – 10 – JHEP05(2018)205 The heavy quark contribution is replaced by the gluon contributions such that  α  X 1 X s fqN = hN | − Gaµν Gaµν N i, mN 12π (3.13) Scenario-II 10-43 = 300 GeV = 300 GeV vS = 5000 GeV M XR MH3 10-45 XENON1t H2017L PandaX - II H2017L 10-47 ΛSX = 0.01 Neutrino limit 10-49 0.002 0.005 0.010 0.020 sin Θ 0.050 Figure 5. DM-Nucleon scattering cross section as a function of sin θ in Scenario-II where we take mX = 300 GeV, mH3 = 300 GeV, vS = 5000 GeV and λSX = 0.5(0.01) for red(blue) line as reference values. The current bounds from XENON1T [32] and PandaX-II [33]. . while collider constraint is less significant compared with direct detection constraints for 2mX > mh [34–36]. Furthermore DM can be produced via heavier Higgs boson H3 if 2mX < mH3 and the possible signature will be mono-jet with missing transverse momentum as pp → H3 j → XXj. However the production cross section will be small when the mixing effect sin θ is small as we assumed in our analysis. Such a process would be tested in future LHC with sufficiently large integrated luminosity while detailed analysis is beyond the scope of this paper. Indirect detection. Here we discuss possibility of indirect detection in our model by estimating thermally averaged cross section in current Universe with micrOMEGAs 4.3.5 using allowed parameter sets from relic density calculations. Since aS aS final state is dominant in scenario-I, we focus on the other scenarios in the following. Figure 6 shows DM annihilation cross section in current Universe as a function of mX where left and right panels correspond to Scenario-II and Scenario-III/IV. In ScenarioII, the cross section is mostly ∼ O(10−26 )cm−3 /s while some points give smaller(larger) values corresponding to the region with 2mX & (.)MH3 as a consequence of resonant effect. The annihilation processes in the scenario provide the SM final state via decay of H3 and {H1 , H ± , A} where H3 decay gives mainly bb̄ via mixing with the SM Higgs and the scalar bosons from second doublet gives leptons. This cross section would be tested via γ-ray observation like Fermi-LAT [37] as well as high energy neutrino search such as IceCube [38, 39], especially when the cross section is enhanced. In Scenario-III, the cross section is mostly ∼ O(10−26 )cm−3 /s and the final states from DM annihilation include – 11 – JHEP05(2018)205 ΣDM-n@cm2D ΛSX = 0.5 Scenario-II Scenario-IIIHIVL 10-25 <Σv>0 @cm3 sD <Σv>0 @cm3 sD 10-25 10-26 10-27 10-26 10-27 10-28 100 200 300 mX @TeVD 400 500 100 200 300 mX @TeVD 400 500 Figure 6. Left: the current DM annihilation cross section in Scenario-II as a function of mX . Right: that for Scenario-III and IV represented by red and blue points. components of Φ1 that are {H1 , H ± , A}. Thus DM mainly annihilate into neutrinos via the decay these scalar bosons while little amount of charged lepton appear from H ± . Therefore constraints from indirect detection is weaker in this scenario. In Scenario-IV, the values of cross section is relatively larger due to the nature of semi-annihilation scenario. In this case final states from DM annihilation give mostly bb̄ via decays of H3 in the final state. Then it would be tested by γ-ray search and neutrino observation as in the scenario-II. 4 Conclusion We consider a neutrino Two Higgs Doublet Model (νTHDM) in which small Dirac neutrino masses are explained by small VEV, v1 ∼ O(1) eV, of Higgs H1 associated with neutrino Yukawa interaction. A global U(1)X symmetry is introduced to forbid seesaw mechanism. The smallness of v1 proportional to soft U(1)X -breaking parameter m212 is technically natural. We extend the model to introduce a scalar dark matter candidate X and scalar S breaking U(1)X symmetry down to discrete Z2 symmetry. Both are charged under U(1)X . The lighter state of X is stable since it is the lightest particle with Z2 odd parity. The soft parameter m212 is replaced by µhSi. The physical Goldstone boson whose dominant component is pseudoscalar part of S is shown to be phenomenologically viable due to small ratio (∼ O(10−9 )) of v1 compared to electroweak scale VEVs of the SM Higgs and S. We study four scenarios depending on dark matter annihilation channels in the early Universe to simplify the analysis of dark matter phenomenology. In Scenario I, Goldstone modes are important. Scenario II is H3 portal. In Scenario III, the dark matter makes use of the portal interaction with Φ1 which generates Dirac neutrino masses. 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International Journal of Management and Applied Research, 2020, Vol. 7, No. 4 A Competitive Analysis of East African Cement Companies using the High Performance Organisation Framework Evans Makini Osano Maastricht School of Management, The Netherlands André de Waal HPO Center, The Netherlands Abstract Sub-standard infrastructure is one of the most significant challenges for economic development in East Africa. Specifically, the availability of affordable, locally produced cement is critical to infrastructural and economic development and job creation in the region. Thus, it is critical for cement companies to achieve better operational and financial results so they can satisfy the need for higher-quality cement for infrastructure projects and to deal with increasing environmental issues. In this article we evaluate, using de Waal’s high performance organisations (HPO) framework, the quality of three cement companies in Kenya and Tanzania to identify improvements these companies can make to their internal organisation and operations so they can achieve better financial and operational results. The research results indicate that whilst all three companies were yet to achieve HPO status, the best performing company with the highest HPO score also had the best financial performance over the past 10 years. The research also resulted in a series of recommendations on the basis of the weaknesses identified in the cement companies. Keywords: High-Performance Organisations; Cement Industry; Kenya; Tanzania; Performance Assessment; HPO Received: 1 May 2020 Revised: 10 August 2020 Accepted: 30 Sept 2020 ISSN 2056-757X https://doi.org/10.18646/2056.74.20-032 A Competitive Analysis of East African Cement Companies using the High Performance Organisation Framework 1. Introduction Sub-standard infrastructure in East Africa has been a major impediment to regional economic development and negatively impacts the quality of life of the residents in the region (Gregory and Sovacool, 2019). A number of studies, notably Kenya Association of Manufacturers (2006), Ministry of Trade and Industry (2007), World Bank (2016) and UNIDO (2001), identified inadequate infrastructure as the most significant challenge for economic development and creating better livelihoods in Kenya and Tanzania. The availability of affordable locally produced cement is critical to cheaper housing, and more infrastructural and economic development and job creation. Cement is a key ingredient in the construction sector, particularly for infrastructure development. Thus, it is critical for cement companies to achieve better operational and financial results so they can satisfy the increasing demand for higher-quality cement for infrastructure projects. At the same time, cement companies have to deal with increasing environmental issues. Cement production consumes large quantities of raw materials and energy and is therefore a significant contributor to climate change, accounting for approximately eight percent of global man-made carbon dioxide (Miller et al., 2018). The study described in this article focuses on evaluating the quality of cement companies so it becomes clear where these organisations can apply improvements in their internal organisation and operations to better deal with the environmental issues while at the same time achieving better financial and operational results. As evaluation tool, de Waal’s HPO Framework (de Waal, 2007, 2012) was used, as this framework is a scientifically validated conceptualization of high performance organisations (HPOs) which highlights the factors that help companies strengthen their internal organisation, thus making them able to achieve higher performance (Do and Mai, 2020). In this respect, the HPO is defined as an organisation that achieves financial and non-financial results that are significantly better than those of its peer group over a period of five years or more, by focusing in a disciplined way on that what really matters to the organisation (de Waal, 2012, p.5). In many previous studies it has been shown that the higher a company scores on the HPO Framework, the better its performance (de Waal, 2012, 2020; de Waal and Goedegebuure, 2017). Therefore the aim of this study was to evaluate the HPO scores of selected cement companies and subsequently compare their internal organisation, operation and culture to identify the differences which make one cement company perform better than the other. As an insufficiently developed infrastructure is a regional problem in East Africa, as a research population cement companies were selected from two neighbouring countries in the region, Kenya and Tanzania. It has to be noted that because of confidentiality reasons: (a) the companies have been made anonymous, and (b) the research took place eight years ago and this description had to wait until the financial results given in this article are no longer representative of the current situation of the case companies (this however in itself does not affect the relevance of the study and the mechanisms studied). The study has both theoretical and practical contributions to make. International Journal of Management and Applied Research, 2020, Vol. 7, No. 4 - 455 - A Competitive Analysis of East African Cement Companies using the High Performance Organisation Framework Theoretically, the research results will add to the still rather limited literature on management, high performance and HPOs in Africa (Areneke et al., 2019). Practically, the research results show that the HPO Framework can be used to improve the regional cement companies; this will offer the opportunity for other African cement companies and potentially also other African manufacturing companies in the region to improve themselves, using the HPO Framework. The remainder of this article is structured as follows. In the next section the cement industry in Kenya and Tanzania are introduced. Subsequently, the HPO Framework is described after which the research approach is given. This is followed by an overview of the research results and analysis thereof. The article ends with a conclusion, the limitations to the research, and opportunities for future research. 2. Study Background This section is divided into two parts. The first part describes the cement industry in Kenya and Tanzania, two countries located in East Africa. The second part discusses the HPO Framework and its application in the African context. 2.1. The cement industry in Kenya and Tanzania Cement industry growth in both Kenya and Tanzania has been strong over the last ten years due to resurgence in the building and construction sectors both locally and in the regional market. Kenya is self-sufficient in cement production with a small surplus that is exported regionally. The cement sector was the second fastest growing in Kenya generating a growth rate of 12.7 percent in 2013. The construction sector accounted for 4.4 percent of Kenya’s GDP in 2013. In 2013, there were six cement companies in Kenya with a combined milling capacity of 7.2 million metric tons per annum. Production stood at 5.1 million tonnes against local consumption of 4.3 million tonnes in 2013. Three cement manufacturing companies were listed on the Nairobi Stock Exchange with a combined market capitalization of US$1.1 billion (Ministry of Trade and Industry, 2014). Tanzania, in contrast to Kenya, was not self-sufficient in cement although planned capacity increases would more than meet the current shortfall. In 2013, cement consumption stood at 3.4 million tons against production of 2.3 million tones with the shortfall met by imports. The construction sector accounted for 6.5 percent of GDP in 2013, with growth rate averaging 9.8 percent between 2004 and 2013. The sector was a beneficiary of increased spending on infrastructure by the Tanzanian government and donor agencies in recent years. The country had three cement companies, two of which were listed on the Dar es Salaam Stock Exchange (Ministry of Industry and Trade, 2014). The research was conducted at three cement companies, two in Kenya and one in Tanzania. These were Company KA and Company KB, both in Kenya, and Company TC in Tanzania. As stated before the companies had to be made anonymous, and International Journal of Management and Applied Research, 2020, Vol. 7, No. 4 - 456 - A Competitive Analysis of East African Cement Companies using the High Performance Organisation Framework although they could still be traceable (as there are not many cement companies in both countries), using synonyms indicates that their current situation cannot and should not be compared to that during the time of the research. The three companies represented at the time of the research fifty percent of the cement companies in Kenya and Tanzania and accounted for over four percent of the cement produced in both countries in 2013. 2.2. High Performance Organisations A High Performance Organisation (HPO) is defined as “an organisation that achieves financial and non-financial results that are exceedingly better than those of its peer group over a period of time of five years or more, by focusing in a disciplined way on that what really matters to the organisation” (De Waal, 2012, p.5). Non-financial results relate to customer satisfaction, customer loyalty, employee loyalty and quality of products and services. As the evaluation mechanism for the study the de Waal HPO Framework was chosen because, as Do and Mai (2020, p. 305) stated, “across the HPO literature, we found only the HPO framework developed by de Waal (2012) as an example of scientifically validated conceptualization of HPO.” Another reason to use the HPO Framework is that is has been applied quite extensively in the African context (de Waal, 2012; de Waal and Chachage, 2011; de Waal and Mulimbika, 2017; de Waal and Olale, 2019; Habyarimana and de Waal, 2020; Honyenuga et al., 2014, 2019). The factors and characteristics of the HPO Framework were determined during a multiyear study consisting of two phases. In the first phase, a literature review of 290 studies into high performance was undertaken to identify common characteristics or themes explaining HPOs (de Waal, 2007). The analysis initially identified 189 potential characteristics which, using a questionnaire distributed to profit and non-profit companies worldwide and subsequent statistical analysis, were reduced to 35 characteristics in five factors. These factors basically make up an HPO and have a direct positive relation with competitive performance (de Waal, 2012). The five factors are Management Quality, Openness and Action Orientation, Longterm Orientation, Continuous Improvement and Renewal, and Employee Quality (see the Appendix for the detailed HPO characteristics). HPO factor Management Quality is primarily focused on leadership, managers being sound role models who nurture and inspire trust, respect and enthusiasm, and are performance-oriented both in their ability to make decisions and to execute them. HPO factor Openness and Action Orientation encourages dialogue and change in the organisation, essential for creating openness, renewal and innovativeness. Openness also fostered innovation by improving the flow and frequency of information implying that promising ideas in R&D, product development, strategy and marketing are aired and refined quickly. HPOs continuously strive for Continuous Improvement and Renewal to create new sources of competitive advantage through new product development, innovation, simplification and improvement in processes and gaining mastery in key competences. Striving for continuous improvement underlies initiatives such as Six Sigma and Total Quality Management. HPO factor Employee Quality requires that an organisation must not only have good people who are energized and focused on achieving extraordinary International Journal of Management and Applied Research, 2020, Vol. 7, No. 4 - 457 - A Competitive Analysis of East African Cement Companies using the High Performance Organisation Framework performance but that they are also able to continuously update their skills. Finally, HPO factor Long-term Orientation ensures that the organisation maintains mutually beneficial enduring long-term relationships and partnerships with stakeholders and is socially responsible. In fact, HPOs are characterized by their corporate social responsibility (CSR) awareness, also referred to as people, profit and planet (de Waal and Orcotoma Escalante, 2008). In this respect, de Waal and Orcotoma Escalante (2008) identified seven CSR aspects related to HPO characteristics and verified these at two mining companies in Peru, demonstrating that the mining company with the strongest financial performance also had the highest CSR scores. An organisation can evaluate its current performance compared to the highperformance level - between 8.5 and 10 on a scale of 1 to 10 (de Waal, 2012) - and identify challenges and possible improvements on the route to high performance by conducting an HPO diagnosis. The HPO diagnosis process starts with management and employees completing the HPO Questionnaire, which consists of statements based on the 35 HPO characteristics. Respondents rate their organisation on a scale of 1 (not at all) to 10 (very much so) on these statements. The individual scores are averaged to provide scores on the HPO factors for the complete organisation. These average scores indicate which HPO factors and characteristics the company needs to improve in order to become an HPO, the organisation drafts improvement actions for these factors and characteristics which then are incorporated in an HPO transformation plan. 3. Research Approach The aim of the research was to compare the cement industry in Kenya and Tanzania, to identify which particular HPO characteristics made a cement company perform strongly and whether there was a difference between these identified characteristics across the two countries. The financially best and worst performing cement companies in each country at the date of the research were asked to participate. The worst performing company in Tanzania declined to participate and therefore was not included in the research. Primary data on the companies was collected by use of the HPO Questionnaire, to determine the overall and individual scores on the five HPO factors and 35 HPO characteristics. Initially the HPO Questionnaire was distributed through the internet; however, the response rate was very low, mainly due to limited internet access for many potential respondents. Consequently, the questionnaire was distributed manually to the participating companies. The lead researcher made field visits to all these locations and undertook the distribution of the questionnaires and interviews with the CEO and other management team members in the companies. The questionnaire was distributed over a stratified sample aimed at targeting the functional areas of the companies including manufacturing, distribution/logistics, marketing and finance, and to both managers and employees. This was achieved through the support of the human resources departments of these organisations. The researcher employed a research assistant to enter the completed questionnaires into the web-based database system. Table 1 provides information on the response per company. International Journal of Management and Applied Research, 2020, Vol. 7, No. 4 - 458 - A Competitive Analysis of East African Cement Companies using the High Performance Organisation Framework Table 1: Information on the participating cement companies Company information Country Number of employees Number of plants Capacity (million tons) Market share (%) Number of questionnaires Number of respondents Response rate (%) Response rate (as % of employees) KA Kenya 1,300 1 3.25 16 45 34 76 3 KB Kenya 700 3 2.90 24 70 60 86 9 TC Tanzania 300 2 3.15 42 50 42 84 14 A total of 165 questionnaires were distributed with a response rate of 82 percentage, split down as 76, 86 and 84 percentage respectively for Company KA, Company KB and Company TC. This response rate was relatively high as the lead researcher was able to solicit the support of the top management in the data collection. The questionnaires were administered in the English language and were not translated into the local languages. The choice of English language was mainly on account of its widespread use in Kenya and Tanzania, particularly in the corporate world. Subsequently, in-depth interviews with managers at the three companies helped to ascertain the reasons for the various HPO scores. At Company KA four management staff in the manufacturing, procurement and human resources departments were interviewed. At Company KB interviews were held with the deputy chief executive at different points in the research process to obtain a better understanding of the industry dynamics and Company KB’s relative strengths and positioning. At Company TC, three management staffs from the human resources and manufacturing departments were interviewed. Organisational performance was determined using export performance and company performance over a period of time. This involved reviewing secondary data from sources such as the National Bureau of Statistics in Kenya and Tanzania, Ministries of Trade and Industry, company specific data (where available) as well as industry data. 4. Research Results and Analysis This section first provides the results of the HPO Questionnaire and the financial results of the companies, and then discusses these results in-depth using information collected during the interviews. 4.1. Scores Table 2 summarizes the HPO scores for the three participating cement companies, while Figure 1 provides a graphical representation of these scores. International Journal of Management and Applied Research, 2020, Vol. 7, No. 4 - 459 - A Competitive Analysis of East African Cement Companies using the High Performance Organisation Framework Table 2: HPO scores for the three East Africa Cement Companies HPO factors Company KA Company KB Company TC Management Quality 6.4 8.3 7.3 Openness and Action Orientation 5.9 7.6 6.6 Long-term Orientation 7.0 8.5 7.7 Continuous Improvement 6.5 8.0 7.5 Employee Quality 6.9 8.2 7.6 Average HPO score 6.6 8.1 7.3 Figure 1: HPO graphs for the three East Africa Cement Companies The financial performances of the three companies over the past 10 years were reviewed, to analyze margins and growth in profitability and shareholder value. As the cement sector is a cyclical industry with a cycle time of 5-8 years, a 10-year period was therefore selected to ensure that performance of the companies did not just reflect a single business cycle. The average change in net profits was a computed as the percentage change in net profits between the financial year ending 2013 and the year ending 2003, divided by 10 (number of years during the period). Market capitalization was used as a proxy for shareholder value. The shareholder value (%) is the change in market capitalization between 2003 and 2013 divided by 10. The results are given in Table 3. International Journal of Management and Applied Research, 2020, Vol. 7, No. 4 - 460 - A Competitive Analysis of East African Cement Companies using the High Performance Organisation Framework Table 3: HPO score and financial performance of cement companies Indicators Company KA Company KB Company TC Average Change in Net Profits p.a. -7% 129% 40% Shareholder Value p.a. 2% 361% 19% HPO Score 6.6 8.1 7.3 Figure 1 shows that the three cement companies share the same HPO profile, that is, the lines in Figure 1 have the same shape. It is thus clear that the three companies operate in the same industry but are of different strengths (i.e. have different HPO scores). Table 3 indicates there is alignment between the HPO scores of the companies and their financial results, i.e. Company KB has the highest HPO score and the best financial results, with Company KA achieving the opposite, and Company TC being in the middle performance-wise. 4.2. General analysis of the HPO scores Maybe characteristically, the interviews at Company KA did not yield much information, as interviewees were not that open. This might not have come as a surprise as Company KA registered poor performance in openness and action orientation. From the interviews it was gathered that frequent management changes particularly at the Chief Executive level - were considered destabilizing to the organisation. It was also felt that decision-making was unduly slow, particularly in regard to non-performers. The slow decision making process may be due to cultural issues. Collectivistic culture such as in Kenyan and Tanzanian societies may slow decision-making as it encourages wide consultations before decisions can be made (Amoako-Agyei, 2009; Hofstede, 1980). And, as Amoako-Agyei (2009) noted, collective solidarity may prevent managers from taking decisive action against nonperformers. Another reason for the slow decision-making process in Company KA is that this is by far the largest organisation of the three cement companies, with more management and therefore decision-making layers. Company KB saw its core competence as its ability to continuously improve and innovate. This enabled the company to reduce production costs, serve new markets and extend its product mix. The company was also willing to take calculated risks. In particular, it was proud of its project management skills which enabled it to complete projects on time at substantially lower costs compared to competitors. Company KB had the highest score on the management quality factor. In particular, the respondents rated the company highly on trust, integrity, confidence and results orientation. However, the management did not perform as well on speed in decision-making, decisiveness with regard to non-performers and holding members accountable on results. This suggests that the performance culture had not necessarily permeated the entire organisation, reflecting significant scope for improvement. Company KB’s management explained that KB was a relatively small company, constrained by resources. They were therefore cautious in making decisions and implementing plans, affecting the speed of making decisions. This cautious, slower decision making might also be due to the nature of the industry, which is highly capital intensive and with International Journal of Management and Applied Research, 2020, Vol. 7, No. 4 - 461 - A Competitive Analysis of East African Cement Companies using the High Performance Organisation Framework high indebtedness/gearing ratios which would make mistakes costly to the company. This could also explain management’s lack of tolerance of mistakes. In theory, this would hinder innovation in the company as making mistakes can be a learning opportunity. Management indicated that innovation was considered more important by the higher management echelons, a feeling which had not been dispersed to other organisational levels. Innovations were mainly around energy efficiency and reduction in the cost of production. Focus was squarely on the production line where line balancing and enhancement were applied to ensure that the over 10,000 pieces of equipment operated optimally. The plant operations were also broken down to small units and energy consumption at each of these units was closely monitored and appropriate switching sequence applied to reduce big load power consumption. The new production line would also take alternative fuels, particularly at the pre-process level, which facilitated greater energy savings. In 2009, the company won the Kenya Institute of Management Energy Innovation Award and its CEO was awarded Africa’s Innovation Leader of the Year Award by the Africa Investor, recognizing the firm’s ability at generating new ideas and successfully executing them. In particular, the company was recognized for reducing the cost of manufacturing and building new plants. Many respondents felt that Company KB did not provide a secure workplace, in the sense of being a safe and healthy place to work, for organisational members. Management explained that it had provided an Employee Share Ownership Program (ESOP) for employees and had relatively low employee turnover. However, it did not for instance run a medical and retirement benefits schemes – which may partly explain the low ranking on this attribute. At Company TC, the interviewees indicated that there was a strong focus on social and environmental management in the company. They also felt that Company TC provided a good working environment for organisational members, and considered the organisation to be performance driven. At the same time, they were of the view that management did not allow for the making of mistakes, which is an essential element in encouraging innovation, which might explain the lower scores compared to Company KB for innovating products and processes. 4.3. Comparative analysis of the HPO factors The biggest difference in scores between the companies is found in HPO factor Management Quality (1.9 points difference between Company KB and Company KA), closely followed by HPO factor Openness and Action Orientation. Management plays an important role in an organisation; it provides leadership, sets the tone and strategy, creates a conducive environment and inspires the organisation to achieve its objectives. Management also attracts, nurtures talent and motivates employees. It sets the vision, organizes the business, sets a good example and creates an environment with the right incentives for achievement of the organisational goals. This is ultimately reflected in the innovations and continuous improvements that take place in the organisation and the commitment to the long-term goals. Management significantly influences the other International Journal of Management and Applied Research, 2020, Vol. 7, No. 4 - 462 - A Competitive Analysis of East African Cement Companies using the High Performance Organisation Framework four characteristics of an HPO and therefore plays a disproportionately large influence in the attainment and maintaining of HPO status. Company KA had the lowest scores of the three companies on the HPO characteristics of Management Quality, which means the respondents of Company KA deemed their management not to be highperformance quality yet, indicating a lot of development was needed in this area. This development of management was going on in the other two companies, thus significantly contributing to the performance of these companies as well-trained management is a success factor for organisations in general (de Waal, 2020) and thus also for African organisations (Douglas et al., 2015; Guo and Bartram, 2019, Tunyia et al., 2019) especially as managerial skills are still in short supply in this area (Barasa et al., 2017). 4.4. Comparative analysis of HPO factor Openness and Action Orientation Openness and action orientation ensures that the good intentions and strategies of management are put into action. By creating an open culture, a healthy dialogue is maintained between management and staff, learning from mistakes is promoted and creativity is nurtured. Action orientation ensures good and timely execution, key in the attainment and maintaining of HPO status. Interestingly, all three companies registered the lowest score on HPO factor Openness and Action Orientation. Further analysis of the results showed that the companies had the lowest scores for the level of dialogue and communication with employees, and for management allowing mistakes. This seems in contradiction with the African culture being highly collectivistic and group-oriented (Hofstede, 1980), thus one would expect that managers in Kenya and Tanzania would be better at dialoguing with employees. There are two possible explanations. First, in a collectivistic culture, “keeping peace” is considered important and people may not be inclined to speak openly, avoiding “rocking the boat”. Secondly, in the context of a culture of relatively high power distance, where superiors wield much power and subordinates are relatively passive, people may not want to question authority. At the same time management does not feel compelled to widely consult with staff. There may be a lot of communication but not necessarily dialogue with managers talking a lot to employees instead of having a real conversation, which encompasses an exchange of ideas and opinions on the basis of which the managers make decisions. 4.5. Comparative analysis of HPO factor Long-term orientation The three companies recorded strong scores on most of the HPO characteristics measuring Long-term Orientation. These can be explained in that the three companies had embraced corporate sustainability in their operations, in the shape of elaborate policies and processes aiming at mitigating their environmental impact, including processes to cut energy consumption, and regeneration and reforestation at disused quarries. They also had support programs, particularly in health and education, for the surrounding communities. Other activities included energy efficiency improvement programs, including alternative fuels. The only exception in regard to the high scores was the tenure of management in the companies. In particular, Company KA had as International Journal of Management and Applied Research, 2020, Vol. 7, No. 4 - 463 - A Competitive Analysis of East African Cement Companies using the High Performance Organisation Framework many as seven chief executives in the previous seven years, an average of one chief executive per year! 4.6. Comparative analysis of HPO factor Employee Quality Regarding HPO factor Employee Quality, Company KB scored the highest, possibly because the company had, of all the companies, the most elaborate internship and training programs for its employees. In 2013, for instance, 81 students drawn largely from the engineering field benefitted from the internship program. Company KB’s internship program played the dual role of empowering the students thus contributing to society and creating a well-filled pipeline of potential new employees. Further, on a yearly basis, at least 20 percent of Company KB’s workforce participated in various training programs. This educational effort is very welcome and needed as in general the service delivery in the educational sector in Kenya (and for that matter also in Tanzania) is rather weak (Cheruiyot and Maru, 2013; Hausken and Ncube, 2018). 4.7. Comparative analysis of the financial results Figure 2 gives the operating profit margins of the three cement companies, for the period 2003 – 2013. We have used operating margin percentage as these can be compared well across the companies because the size differences of companies are equalized by the percentages. Figure 2: Operating profit percentage for the three cement companies Company KB’s operating profit margin improved from 9.0 percent in 2003 to 16.9 percent in 2013, caused by continuous improvements in processes and new International Journal of Management and Applied Research, 2020, Vol. 7, No. 4 - 464 - A Competitive Analysis of East African Cement Companies using the High Performance Organisation Framework investments in a state of the art cement plant, diversification into higher margin cement businesses, better cost control, and operational efficiency improvements in the processes. Over a 10-year period the operating margin percentage has steadily improved, reflecting the robust nature of the company. Company KB was upbeat about its financial prospects for the next ten year period. The financial performance of Company KA over the previous ten years had been mixed with a clear downward trend, as Figure 2 clearly shows. This was partly caused by foreign exchange losses and high operating costs especially in staff costs. For instance, in 2012 staff costs constituted 12 percent of the cost of manufacturing compared to a 5-6 percent average for the other cement companies in the region. Company KA was pessimistic about its financial prospects for the next ten year period as it saw many threats, especially from foreign competitors, potentially hurting its future business and results. The financial performance of Company TC was exceptional, although erratic, over the ten year period in question. The operating margin pattern can be favourably attributed to cement price increases and better cost control, and unfavourably to higher costs caused by the company relying on expensive imported clinker following the upgrade in its milling capacity. Company TC was upbeat about its financial prospects for the next ten year period. 4.8. Recommendations Following the analysis in the previous sections, the following recommendations can be made to the three cement companies to improve or maintain their HPO scores:  Management Quality – The companies need to design management development plans to improve the skills, behaviours and performance of its managers. It is particularly critical for Company KA which recorded the lowest scores on the HPO factor on management quality. The development plans should especially focus on the development of behaviours in the following areas: decision making and action taking, and dialoguing.  Openness and Action Orientation – The companies should encourage more openness, including improving the level of dialogue and communication with the employees. In particular, management should be trained to improve its dialoguing and communication skills.  Long-term Orientation - The companies should invest more time and resources in making their organisations a secure work place for their employees. Specific actions needed include improvements in corporate governance in Company KA where there have been too frequent changes in the top management. Further, Company KB should consider introducing medical and retirement schemes for its employees.  Continuous Improvement and Renewal - All three companies should focus on simplifying and aligning their organisation processes in order to improve their performance.  Employee Quality - The three companies have to deal with different issues to improve the quality of their workforce. Company KB and Company KA should make employees accountable for their results. This requires the design and implementation of an appropriate performance management program. In addition, International Journal of Management and Applied Research, 2020, Vol. 7, No. 4 - 465 - A Competitive Analysis of East African Cement Companies using the High Performance Organisation Framework Company KA’s management needs to inspire its employees to achieve extraordinary results. For Company TC, the focus should be on training and cultural change programs for their employees to become more resilient and flexible. 5. Conclusion The study described in this article set out to evaluate the quality of cement companies in Kenya and Tanzania. The goal was to identify the current strengths and weaknesses of the three companies, so it would become clear where these organisations could apply improvements in their internal organisation and operations to achieve better financial and operational results. As the evaluation tool, de Waal’s HPO Framework (de Waal, 2007, 2012) was used. This provided another research goal, namely to evaluate whether there was a relation between the HPO scores and the financial results of the companies. The research results indicate that whilst all three companies were yet to achieve HPO status, Company KB was the best performer with an HPO score close to the HPO status and consistent strong financial performance over the past 10 years. The research also resulted in a series of recommendations on the basis of the weaknesses identified. There were some limitations to the study. As the cement industry in both Kenia and Tanzania was dominated by a few firms, there was a limited number of potential companies in which to carry out the research. For instance, there were only three cement manufacturing firms in Kenya and three in Tanzania. Future research could expand to the cement industry in other countries, both in Africa and other parts of the world. In addition, the research could be expanded to other industries in Kenya and Tanzania, to possibly achieve generalization of the research findings. Another limitation was that the findings were based on only the managers’ views, future research could and should include the views of employees, to get a better and less biased balance in the research results. Also, the research did not look into the effects of the recommendations, i.e. whether cement companies implementing the recommendations would actually perform better. Future longitudinal research could look into that. Future comparative study could go more in-depth in the cement companies themselves, to evaluate whether there are HPO score and financial performance differences between plants and branches. Finally, to evaluate possible cultural differences, it would be useful to replicate the study in West Africa and Southern Africa. 6. References 1. Amoako-Agyei E. 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Kenya Association of Manufacturers (2006), Manufacturing in Kenya: Survey of Kenya’s Manufacturing Sector, Kenya Association of Manufacturers, Nairobi. 23. Miller, S. A.; John, V. M.; Pacca, S. A. and Horvath, A. (2018), “Carbon dioxide reduction potential in the global cement industry by 2050”, Cement and Concrete Research, Vol. 114, pp. 115-124. https://doi.org/10.1016/j.cemconres.2017.08.026 24. Ministry of Industry and Trade (2014), Quarterly Economic Bulletin 2014, Dar Es Salaam: Ministry of Industry and Trade 25. Ministry of Trade and Industry (2007), Master Plan for Kenyan Industrial Development, Nairobi: Ministry of Trade and Industry,. 26. Ministry of Trade and Industry (2014), Economic Survey 2014, Ministry of Trade and Industry, Government of Kenya, Nairobi. International Journal of Management and Applied Research, 2020, Vol. 7, No. 4 - 468 - A Competitive Analysis of East African Cement Companies using the High Performance Organisation Framework 27. Tunyi, A.A., Agyei-Boapeah, H., Areneke, G. and Agyemang, J. (2019), “Internal capabilities, national governance and performance in African firms”, Research in International Business and Finance, Vol. 50, pp. 18-37. https://doi.org/10.1016/j.ribaf.2019.04.009 28. World Bank (2016), Infrastructure for Economic Growth and Shared Prosperity in Kenya: Addressing Infrastructure Constraints, Promoting Economic Growth, and Reducing Inequality, [Online] Available from: https://www.worldbank.org/en/results/2016/07/21/infrastructure-for-economicgrowth-and-shared-prosperity-in-kenya-addressing-infrastructure-constraintspromoting-economic-growth-and-reducing-inequality [Accessed on 14 May 2020]. 29. United Nations Industrial Development Organisation (UNIDO) (2001), Tanzania – Sustainable Industrial Development and Competitiveness (Analyzing Competitiveness, Strategies, Policies and Action Plan to Accelerate Industrial Development), Austria: UNIDO. Appendix This Appendix provides the detailed scores on the HPOP characteristics for the three participating cement companies KA, KB and TC. HPO FACTORS Continuous Improvement and Renewal 1. The organisation has adopted a strategy that sets it clearly apart from other organisations 2. In the organisation processes are continuously improved. 3. In the organisation processes are continuously simplified. 4. In the organisation processes are continuously aligned. 5. In the organisation everything that matters to performance is explicitly reported. 6. In the organisation both financial and non-financial information is reported to organisational members. 7. The organisation continuously innovates its core competencies. 8. The organisation continuously innovates its products, processes and services. Openness and Action Orientation 9. Management frequently engages in a dialogue with employees. 10. Organisational members spend much time on communication, knowledge exchange and learning. 11. Organisational members are always involved in important processes. 12. Management allows making mistakes. 13. Management welcomes change. 14. The organisation is performance driven. KA KB TC 6.4 8.0 7.4 7.1 6.2 5.9 8.1 7.3 7.8 8.3 7.3 7.2 6.8 8.3 7.5 6.3 7.9 7.4 6.2 8.1 7.3 6.1 8.3 7.5 5.6 7.9 6.9 5.1 7.1 6.6 6.0 8.0 7.1 4.6 6.4 7.0 5.9 8.3 8.5 3.9 7.4 7.9 International Journal of Management and Applied Research, 2020, Vol. 7, No. 4 - 469 - A Competitive Analysis of East African Cement Companies using the High Performance Organisation Framework Management Quality 15. Management is trusted by organisational members. 16. Management has integrity. 17. Management is a role model for organisational members. 18. Management applies fast decision making. 19. Management applies fast action taking. 20. Management coaches organisational members to achieve better results. 21. Management focuses on achieving results. 22. Management is very effective. 23. Management applies strong leadership. 24. Management is confident. 25. Management is decisive with regard to non-performers. Employee Quality 26. Management always holds organisational members responsible for their results. 27. Management inspires organisational members to accomplish extraordinary results. 28. Organisational members are trained to be resilient and flexible. 29. The organisation has a diverse and complementary workforce. Long-Term Commitment 30. The organisation maintains good and long-term relationships with all stakeholders. 31. The organisation is aimed at servicing the customers as best as possible. 32. The organisation grows through partnerships with suppliers and/or customers. 33. Management has been with the company for a long time. 34. The organisation is a secure workplace for organisational members. 35. New management is promoted from within the organisation. 6.2 6.4 6.3 5.4 5.5 8.6 8.8 8.2 8.3 7.9 7.1 7.5 7.2 6.9 6.8 6.4 8.0 7.6 7.3 6.3 6.5 6.8 5.3 8.6 8.2 8.3 8.9 7.3 7.9 7.3 7.2 7.3 6.6 6.5 8.1 8.0 6.2 7.8 7.6 5.9 7.4 8.0 8.3 7.2 7.2 7.6 8.6 7.8 7.5 8.8 8.1 7.9 8.8 8.3 5.9 5.1 7.0 8.7 7.6 8.4 7.3 7.1 7.5 International Journal of Management and Applied Research, 2020, Vol. 7, No. 4 - 470 - 

W3112593059.txt

PLOS ONE RESEARCH ARTICLE Reduction of oxidative stress on DNA and RNA in obese patients after Roux-en-Y gastric bypass surgery—An observational cohort study of changes in urinary markers Elin Rebecka Carlsson ID1,2*, Mogens Fenger1, Trine Henriksen3, Laura Kofoed Kjaer3, Dorte Worm4, Dorte Lindqvist Hansen5, Sten Madsbad6, Henrik Enghusen Poulsen ID3* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Clinical Biochemistry, Copenhagen University Hospital Hvidovre, Hvidovre, Denmark, 2 Department of Clinical Biochemistry, Nordsjaellands Hospital, University of Copenhagen, Hilleroed, Denmark, 3 Department of Clinical Pharmacology, Bispebjerg Frederiksberg Hospital, Copenhagen University Hospital, Copenhagen, Denmark, 4 Department of Medicine, Amager hospital, Copenhagen, Denmark, 5 Steno Diabetes Center Copenhagen, Gentofte, Denmark, 6 Department of Endocrinology, Copenhagen University Hospital Hvidovre, Hvidovre, Denmark * elin.rebecka.carlsson@regionh.dk (ERC); henrik.enghusen.poulsen.01@regionh.dk (HEP) OPEN ACCESS Citation: Carlsson ER, Fenger M, Henriksen T, Kjaer LK, Worm D, Hansen DL, et al. (2020) Reduction of oxidative stress on DNA and RNA in obese patients after Roux-en-Y gastric bypass surgery—An observational cohort study of changes in urinary markers. PLoS ONE 15(12): e0243918. https://doi.org/10.1371/journal. pone.0243918 Editor: Yvonne Böttcher, University of Oslo, NORWAY Received: April 17, 2020 Accepted: November 18, 2020 Published: December 14, 2020 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0243918 Copyright: © 2020 Carlsson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Increased oxidative stress in obesity and diabetes is associated with morbidity and mortality risks. Levels of oxidative damage to DNA and RNA can be estimated through measurement of 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8oxoGuo) in urine. Both markers have been associated with type 2 diabetes, where especially 8-oxoGuo is prognostic for mortality risk. We hypothesized that Roux-en-Y gastric bypass (RYGB) surgery that has considerable effects on bodyweight, hyperglycemia and mortality, might be working through mechanisms that reduce oxidative stress, thereby reducing levels of the urinary markers. We used liquid chromatography coupled with tandem mass spectrometry to analyze the content of 8-oxodG and 8-oxoGuo in urinary samples from 356 obese patients treated with the RYGB-procedure. Mean age (SD) was 44.2 (9.6) years, BMI was 42.1 (5.6) kg/m2. Ninety-six (27%) of the patients had type 2 diabetes. Excretion levels of each marker before and after surgery were compared as estimates of the total 24-hour excretion, using a model based on glomerular filtration rate (calculated from cystatin C, age, height and weight), plasma- and urinary creatinine. The excretion of 8oxodG increased in the first months after RYGB. For 8-oxoGuo, a gradual decrease was seen. Two years after RYGB and a mean weight loss of 35 kg, decreased hyperglycemia and insulin resistance, excretion levels of both markers were reduced by approximately 12% (P < 0.001). For both markers, mean excretion levels were about 30% lower in the female subgroup (P < 0.0001). Also, in this subgroup, excretion of 8-oxodG was significantly lower in patients with than without diabetes. We conclude, that oxidative damage to nucleic acids, reflected in the excretion of 8-oxodG and 8-oxoGuo, had decreased significantly two years after RYGB—indicating that reduced oxidative stress could be contributing to the many long-term benefits of RYGB-surgery in obesity and type 2 diabetes. Data Availability Statement: Data were extracted from hospital repositories and contain potentially PLOS ONE | https://doi.org/10.1371/journal.pone.0243918 December 14, 2020 1 / 19 PLOS ONE identifying or sensitive patient information that, due to local regulations and current legislation, cannot be shared publicly. Data requests may be sent either directly to the authors or to the department of Clinical Biochemistry, Copenhagen University Hospital Hvidovre (contact via kliniskbiokemi. hvidovrehospital@regionh.dk or at +45 38 62 11 00). Funding: The collection of samples to the biobank used in this study was partially funded by The Ministry of Higher Education and Science (the UNIK project). The authors received no specific funding for their work related to this study. Competing interests: The authors have declared that no competing interests exist. Gastric bypass surgery and urinary markers of DNA/RNA oxidation Introduction Obesity increases the risk of progression to metabolic syndrome, type 2 diabetes, cardiovascular- and liver disease, as well as certain cancers and is generally associated with an increased all-cause mortality rate [1, 2]. A combination of increased inflammation and oxidative stress, induced by obesity, is suggested to be causative [2–4]. Oxidative stress, broadly defined as an imbalance in the body’s naturally occurring oxidation- and reduction processes which leads to a net increase in concentrations of highly reactive oxygen species (ROS) [5], further seems to accelerate the development of micro- and macrovascular complications in patients with diabetes [6]. Oxidative stress can damage important cell-structures like biological membranes, proteins, lipids, DNA and RNA. Under normal conditions, damage caused by ROS are kept at a minimum through the acts of the body’s own defense system [5]. The products of the repair-mechanisms controlling oxidative damage to nucleic acids can be assessed in urine as the markers 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodG) [7] and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) [8], respectively. 8-oxodG is the result of oxidation of the DNA guanine moiety and 8-oxoGuo is the result of oxidation of the RNA guanine moiety [7]; they are produced when 8-hydroxylation of guanine (the most vulnerable of nucleobases [5]) is repaired or degraded in DNA or RNA, respectively [8], and technically they can be differentiated due to the difference in the ribose and the deoxyribose part of the molecule [8]. Guanine oxidation is known to destabilize the guanine-quadruplexes (so called G4’s) established in the single-strand stretches of promoters (thereby affecting gene transcription) and telomeres [9]. Up to half of the oxidized and damaged DNA is thought to originate from the telomeres [10], suggesting that oxidative stress has an important impact on the ageing process. The exact oxidative mechanism is not clear as several pathways are possible, including hydroxyl radical oxidation [11]. Both 8-oxodG and 8-oxoGuo have shown to be associated with metabolic diseases, including type 2 diabetes [8, 12, 13] and excretion of both 8-oxodG [14] and 8-oxoGuo [15] has been reported increased in obese populations compared to controls. In type 2 diabetes, 8-oxoGuo excretion is higher in patients with than without complications [16] and is prognostic, perhaps even predictive, of mortality-risk and risk of death from macrovascular complications [17, 18]. Little is known about the effect of weight loss on oxidative damage on DNA and RNA. In mice, a recent study found a reduction in DNA damage (assessed with the comet assay) after weight loss in several inner organs [19]. In humans, the same method has been used to show a reduction in lymphocyte DNA damage after weight loss induced by metabolic surgery, in 56 patients twelve months post-operative [20]. The same research group later confirmed its results in vivo, with micronucleus assessment in a similar patient cohort (the type of metabolic surgery performed was not specified in either of the manuscripts) [21]. Nevertheless, these studies together with a report of decreased levels of urinary 8-oxodG, twelve months after laparoscopic sleeve gastrectomy in twenty-one morbidly obese patients [14], indicate that weight loss and/or metabolic surgery might be effective in reducing DNA oxidation. In the latter study, urinary 8-oxodG levels fell gradually to around half the rate twelve months after sleeve gastrectomy, a level that was close to the control group of hundred healthy, non-obese volunteers [14]. The RNA marker 8-oxoGuo was not measured. Roux-en-Y Gastric bypass (RYGB) surgery has a high success-rate in reducing weight, normalizing dyslipidemia and lowering hyperglycemia in both the short- and the long-term [22, 23]. Furthermore, RYGB was proven able to reduce mortality in obese patients with and without diabetes [24]. It is still unknown, if some of these effects are facilitated or sustained through a reduction in oxidative stress. Most of the studies investigating one or several different markers of oxidative stress and antioxidant defense after RYGB in obese patients suggest an PLOS ONE | https://doi.org/10.1371/journal.pone.0243918 December 14, 2020 2 / 19 PLOS ONE Gastric bypass surgery and urinary markers of DNA/RNA oxidation improvement of oxidative stress after the surgery, although results are partly conflicting—for instance some of the studied markers (glutathione, superoxide dismutase and catalase) have been reported to be increased after RYGB in some of the studies and to be decreased in other studies [25–31]. How levels of oxidative DNA and RNA damage, reflected in the urinary markers 8-oxodG and 8-oxoGuo, respectively, are affected by RYGB, has however not been reported, except for a study on obese Zucker rats [32]. This paper reports our findings from a study in a large cohort of 356 patients, where we aimed to examine if a reduction in oxidative stress level (reflected in the levels of the urinary markers 8-oxodG and 8-oxoGuo) could be detected within the first two years after RYGB-surgery. We also aimed to find out if there were differences in the levels of the urinary markers between patient subgroups based on diabetes status and also, if levels differed before or after surgery depending on the outcome after surgery on the diabetic condition. Finally, we aimed to investigate whether there were associations between changes in the oxidative stress markers and other beneficial changes (for example weight loss and improvements in glucose- and lipid metabolism) after RYGB. We hypothesized a priori, that because the urinary markers 8-oxodG and 8-oxoGuo represent an indirect measure of oxidative damage to nucleic acids that is increased in obesity and type 2 diabetes, one would expect a decrease in levels of the two urinary markers after RYGBsurgery, along with the reduction in weight, hyperglycemia and insulin resistance. We also hypothesized that levels of the urinary markers would be higher in patients with type 2 diabetes and possibly, correlated to weight change and/or markers of glucose- and lipid metabolism. Materials and methods Research population The research population is a cohort of patients, mainly of Caucasian ethnicity, who had surgery for obesity with the RYGB procedure between November 2010 and September 2013 and attended pre-examinations and postoperative medical follow-up at Copenhagen University Hospital Hvidovre, Denmark [33]. The criteria for inclusion were a BMI above 35 together with one or several comorbidities related to obesity (type 2 diabetes, resistant hypertension, sleep apnea, infertility in women with polycystic ovary syndrome, lower extremity arthrosis), or a BMI above 50 without complications. Minimum age was 25 years. Exclusion criteria were, apart from general contraindications for surgery and/or anesthesia, severe psychiatric disorder, eating disorder or substance abuse. All patients were instructed to take vitamin and mineral supplementations according to international nutritional recommendations and common practice [34]. Unable- or unwillingness to follow the nutritional recommendations was an exclusion-criteria. As illustrated in S1 Fig, we have included patients who between October 2010 and September 2015, before as well as on minimum one occasion after their RYGB-operation, had delivered urine and blood samples to a research biobank. A total of 356 out of 786 RYGB-operated patients were included, for whom collective data from 1) the urinary analysis, 2) measurement of cystatin C in a matching blood sample and 3) timely recorded data on height, weight and routinely measured plasma creatinine enabled calculation of pre- and postoperative 24-hour 8-oxoGuo and 8-oxodG excretion, as described in the section below. Altogether, 24-hour estimates of 8-oxoGuo and/or 8-oxodG excretion could be calculated in 1254 urine samples, where 356 samples (from 356 patients) were collected before surgery, and 269, 232, 229 and 168 samples were collected at planned intervals of three, six, twelve and twenty-four months after RYGB, respectively, as shown in Table 1. For sixty-nine of the patients, a 24-hour excretion estimate could be calculated for 8-oxoGuo at all four timepoints after RYGB, and for 118, PLOS ONE | https://doi.org/10.1371/journal.pone.0243918 December 14, 2020 3 / 19 PLOS ONE Gastric bypass surgery and urinary markers of DNA/RNA oxidation Table 1. Adherence of the 356 included patients to post-operative follow up (example for 8-oxoGuo). Follow-up combination Number of patients preoperative sample Postoperative samples Samples per patient Number of patients 3 months 6 months 12 months 24 months 1 69 1 1 1 1 1 5 69 2 16 1 1 1 0 1 4 117 3 21 1 1 0 1 1 4 20 1 0 1 1 1 5 60 1 1 1 1 0 6 8 1 1 0 0 1 3 101 7 14 1 0 0 1 1 8 9 1 0 1 0 1 9 22 1 1 0 1 0 10 10 1 0 1 1 0 11 38 1 1 1 0 0 12 11 1 0 0 0 1 2 69 13 13 1 0 0 1 0 14 10 1 0 1 0 0 15 35 1 1 0 0 0 Total 356 356 269 232 229 168 356 https://doi.org/10.1371/journal.pone.0243918.t001 100 and sixty-nine, it could be calculated at three, two and one timepoint postoperative, respectively (Table 1). Twelve months or later after RYGB, follow up excretion estimates could be calculated for 273 (76%) of the included patients. For 8-oxodG, numbers were similar. On average, the preoperative samples were collected 4.56 (4.19–4.93) months (mean with a 95% CI) before surgery. The postoperative samples were collected 3.12 (3.07–3.17), 6.38 (6.27– 6.49), 12.31 (12.16–12.46) and 24.58 (24.28–24.88) months after RYGB surgery, respectively. All urine and blood samples were frozen shortly after sampling at −80˚C and stored between three and eight years at the time for analysis of 8-oxodG and 8-oxoGuo. Clinical data like for example surgery date, bodyweight, blood pressure, as well as smoking status and information about anti-diabetic, anti-hypertensive and lipid-lowering treatment, had been collected in a database at pre- and postoperative consultations and were later validated by cross-check in medical journals and records of prescribed medicine. Information about gender was extracted from the Danish social security number given to every resident in Denmark. Biochemical data from 2009 and onwards, including results from routine bloodand urine analyses, were retrieved from the laboratory information system. In this paper, we have included biochemical data until May 2017. Depending on time for enrolment and on variation in adhesion to postoperative follow-up, some of the patient data records, either clinical and/or biochemical were not complete for all patients. This study was performed in accordance with the Helsinki Declaration and was approved by the scientific Ethics Committee of the Capital Region, Denmark, protocols number HD2009-78 and H-6-2014-029, and by the Danish Data Protection Agency. All participants in the study gave written informed consent. Diabetes subgroups Biochemical data, together with data on antidiabetic medication, were used to divide patients in subgroups according to diabetes status, as illustrated in Fig 1. Diabetes was defined as presence in biochemical records of hyperglycemia (HbA1c > 48 mmol/mol (6.5%), plasma PLOS ONE | https://doi.org/10.1371/journal.pone.0243918 December 14, 2020 4 / 19 PLOS ONE Gastric bypass surgery and urinary markers of DNA/RNA oxidation Fig 1. Subdivision according to diabetes status. Marked with grey color are the groups discussed in this paper. NDH, patients without diabetes; NDH, patients with type 2 diabetes; DH-NDH, patients with diabetes who obtained remission in diabetes after RYGB; DH-DH, patients with diabetes who did not obtain remission in diabetes after RYGB. Twenty-three patients did not fit in any of the groups NDH, DH-NDH or DH-DH. https://doi.org/10.1371/journal.pone.0243918.g001 glucose > 7.0 mmol in the fasting state or > 11.1 mmol as 120 minutes-value in a 75 g oral glucose tolerance test). Remission of diabetes after RYGB was recognized as described previously [35] in patients with diabetes who after RYGB were off antidiabetic medicine, with HbA1c staying below 48 mmol/mol for the entire remaining span of their biochemical records. In tables and figures, the subgroup of patients with diabetes is called DH (short for diabetes and hyperglycemia). NDH is the group without diabetes. The group of patients who obtained remission is called DH-NDH. The patients in group DH-DH did not obtain remission of diabetes. Of the 356 patients, 337 had biochemical data both before and after surgery, ninety-six (27%) of these had type 2 diabetes. Sixty-one (64%) of the patients with type 2 diabetes passed the criteria for remission of diabetes after RYGB, described above. Twenty-three of the patients, four of which had type 2 diabetes, did not fit in any of the three groups NDH, DH-NDH, DH-DH (Fig 1). Analysis of 8-oxodG and 8-oxoGuo The analytical method for measuring concentrations of the urinary markers of nucleic acid damage—liquid chromatography coupled with tandem mass spectrometry—has been described in detail [36]. Half of the samples were analyzed in 2015–2016 and the rest in 2018. To be sure that levels were comparable, analyses of 310 random samples analyzed in 2015– 2016 were repeated in 2018. Mean coefficient of variation (CV) was 14% and 13%, for 8-oxodG and 8-oxoGuo, respectively, when comparing 2018- to 2015–2016 levels. In samples analyzed 2018, urinary concentrations of 8-oxodG and 8-oxoGuo were approximately 4–5 nmol/L and 6 mmol/L higher, respectively. Samples analyzed in 2015–2016, compared to samples analyzed in 2018, had a similar distribution of preoperative and postoperative samples. Both in 2015–2016 and in 2018, analyses have been stable with a day to day CV of less than 8% PLOS ONE | https://doi.org/10.1371/journal.pone.0243918 December 14, 2020 5 / 19 PLOS ONE Gastric bypass surgery and urinary markers of DNA/RNA oxidation and a mean CV at 4–5% for double estimations. Only three results for 8-oxodG (none for 8-oxoGuo) could not be reported due to interference in the chromatograms. Traditionally, to reduce variability of analyte concentration in spot urine samples from variation in urine volume, concentrations of 8-oxodG and 8-oxoGuo were normalized to urinary creatinine concentrations. We analyzed urine creatinine with an in-house assay (Jaffé-method [37]) that has a day to day CV of 3.1%, a CV at 2.8% for double estimations and a trueness at 114% corresponding to the commercial assays (assessed through external quality control). The in-house procedure is half-automatized, using a Biomek robot from Beckman Coulter for sample preparation on a 96-well microplate. The absorbance is measured with a Multiskan FC Microplate Photometer from Thermo Scientific. Creatinine concentrations measured in 218 preoperative urine samples were seen to correlate well to the results from a Cobas 6000 from Roche, measured on the day of sampling, with R2 = 0.96 and a mean CV at 4.9%. Urinary creatinine alone did not, however, suffice as normalization factor in this study, the reasons are explained in the section below. Using a physiological model to adjust for changes in muscle mass. Apart from a large loss of fat mass, weight loss after RYGB includes a substantial decrease (12–16%) in muscle mass [38]. A similar decrease is seen in the excretion rate of urinary creatinine [39]. Decreases in 24-hour urinary creatinine with 18–24% at six and twelve months postoperative follow up after RYGB, compared to preoperative mean values of 1.3 g, have been reported [40, 41]. In a subsample of nineteen patients from our own RYGB-cohort, lean limb mass (a surrogate measure of skeletal muscle mass) was reduced with 11.4% six months after RYGB [42]. The changes in muscle mass after RYGB and the assumed variation they impose on urinary creatinine in our study population challenge the traditional normalization-approach with urinary creatinine mentioned in the previous section, as excretion of creatinine is not constant over time in these patients. To face this challenge, we used a physiological model to calculate estimates of 24-hour excretion of the urinary markers—an approach that has been described in detail and validated in a recent study [43]–for all pre- and postoperative samples. To calculate estimates of GFR, we used the CKD-EPI Cystatin C equation from 2012 in combination with the common formula by Du Bois & Du Bois for calculation of body surface area (BSA). Serum cystatin C was measured on a Cobas 6000 c 501 module (Tina-quant Cystatin C immunoturbidimetric method), Roche Diagnostics. The cystatin C based GFR-estimate was unaffected by weight loss (and hence muscle mass) in the above mentioned study on nineteen RYGB patients from our own cohort [42]. Other biochemical variables All other blood-, plasma- and urine parameters were routine biochemical analyses, measured as described previously [33]. Routine eGFR was calculated on the day of sampling with the CKD-EPI Creatinine equation from 2009. Results reported as > 90 mL/min in the laboratory data system were counted as 90 mL/min. Estimations of insulin resistance according to the Homeostasis Model Assessment (HOMA-IR) were performed in the HOMA2-calculator software, version 2.2.3, using concentrations of Plasma-glucose and C-peptide from the same time of sampling. Due to a systematical bias in Scandinavian laboratories (including ours) measuring HbA1c with high pressure liquid chromatographic methods, HbA1c-results from before January 2013 were adjusted with– 2.7 mmol/mol (0.25%) as previously described [35] to allow for comparisons of levels on both sides of the calibrator change. The NGSP converter converted HbA1cvalues in mmol/mol to %. PLOS ONE | https://doi.org/10.1371/journal.pone.0243918 December 14, 2020 6 / 19 PLOS ONE Gastric bypass surgery and urinary markers of DNA/RNA oxidation Data analysis and statistics Based on previous controlled trials with power calculations [44] we evaluated that the study could be conducted with high power and thus acceptable statistical certainty. All differences, both within the same group (comparisons of pre- and postoperative levels) and between groups have been calculated post hoc to have a power above 0.9 when effect-sizes exceeds 0.8. Estimates of pre- and postoperative 24-hour 8-oxoGuo and 8-oxodG excretion were calculated as described above. Normal distribution, evaluated by inspection of a q-q plot, was seen for most variables, which with few exceptions allowed for the use of parametric statistics. The two urinary markers showed a slight tendency, similar in subgroups, towards light left skewed distributions. The skew was not large enough, however, to abandon parametric statistics. A repeated measures ANOVA combined with Mauchly’s test of sphericity was used, with group mean substitution for missing values (528 out of 1780 datapoints for 8-oxodG and 526 for 8-oxoGuo were imputed); followed by a Greenhouse-Geisser correction for violation of sphericity. Post-operative results were compared with the pre-operative with planned contrast analyses. To be sure not to over-interpret post-operative changes, a Bonferroni correction was applied. Extreme values (identified by inspection of a boxplot) were few and removing them did not alter significance, just as correction for the reduced degrees of freedom due to imputation did not. Because of the unequal gender distribution in our subgroups and because we know, since long, that gender has an influence on the urinary marker levels [45], data from female and male patients were analyzed separately. In the larger female population, differences between patients with and without diabetes and between diabetes subgroups were detected using unpaired t-tests and one-way ANOVA with contrast analysis, respectively. Smoking and lipid-lowering treatment are other possible confounders [46]. To exclude tentative changes in smoking status or in lipid-lowering drug intake during the time of follow up as a reason for a change in urinary marker levels, we also choose to evaluate possible differences between smokers and non-smokers as well as patients on and not on lipid-lowering drugs, by visual interpretation of graph. Correlations between the urinary markers and other variables (including the urinary markers normalized in a traditional manner to urinary creatinine) were investigated with Spearman’s correlation. Statistic calculations were performed in the IBM SPSS Statistics software, versions 22 and 25. Results Preoperative characteristics The mean age in the study population was 44.2 years, BMI was 42.1 kg/m2 and 69.1% of the patients were females. Compared to patients without diabetes, patients with type 2 diabetes were older, with a slightly lower BMI and to a larger part male. A selection of clinical variables is presented below, in Table 2 for patients without diabetes and patients belonging to either of the two main diabetes subgroups discussed in this paper (N = 337). Detailed preoperative clinical and biochemical characteristics are reported in S1 Table for the entire research population (N = 356). Here, it is shown that patients with diabetes had lower cholesterol-levels (consistent with the high number taking cholesterol-lowering drugs) than the patients without diabetes. BSA, cystatin C and estimates of kidney function (eGFR Creatinine and eGFR Cystatin C) were comparable between patients with and without diabetes and between diabetes subgroups, PLOS ONE | https://doi.org/10.1371/journal.pone.0243918 December 14, 2020 7 / 19 PLOS ONE Gastric bypass surgery and urinary markers of DNA/RNA oxidation Table 2. Preoperative characteristics for the three main study population subgroups. ANOVA Pvalue DH-NDH Patients with type 2 diabetes, in remission after RYGB DH-DH Patients with type 2 diabetes with persisting hyperglycemia after RYGB Number of patients in the 241 group (N) 61 31 Age (years) 41.7 (9.1) 49.1 (9.1) 50.8 (6.9) Females (%) 75.5 54.0 55.0 0.003 Bodyweight (kg) 124.9 (22.5) 125.9 (22.6) 115.8 (17.6) 0.078 BMI (kg/m2) 42.7 (5.6) 41.8 (5.9) 40.0 (3.9) 0.029 Present smoker (%) 17.5 22.9 6.5 0.044 Lipid-lowering treatment (%) 10.0 51.0 77.0 < 0.0001 NDH Patients without diabetes < 0.0001 Data are reported as Mean (SD), except for the parameters reported as a percentage or number. Age is on the day of Roux-en-Y gastric bypass (RYGB) surgery. Data represent the closest available data point before surgery. NDH, patients with biochemical glucose markers below diagnostic threshold for diabetes and not on antidiabetic treatment; DH-NDH, patients who obtained remission of diabetes after RYGB; DH-DH, patients who did not obtain remission in diabetes after RYGB. https://doi.org/10.1371/journal.pone.0243918.t002 and all patient groups had a mean blood pressure within the normal range (consistent with an overall high prevalence of anti-hypertensive treatment). Preoperative excretion of 8-oxodG and 8-oxoGuo Mean 24-hour excretion levels of 8-oxodG and 8-oxoGuo, in urine sampled before surgery are reported in detail in Table 3, for both genders and patients with and without type 2 diabetes. In brief, excretion of 8-oxoGuo was higher than excretion of 8-oxodG. There was a highly significant difference between female and male patients, male patients having 40–50% higher excretion levels. In the female subpopulation, 24-hour excretion of 8-oxodG was marginally lower in patients with diabetes than in patients without diabetes, while no difference was seen between patients with and without diabetes for 8-oxoGuo. Table 3. Preoperative excretion of 8-oxodG and 8-oxoGuo for female and male patients with and without diabetes. preoperative 24-hour 8-oxodG (nmol) all NDH DH DH vs. NDH n mean (95% CI) n mean (95% CI) n mean (95% CI) P All 356 18.9 (18.0–19.7) 241 19.1 (18.1–20.1) 96 18.5 (16.7–20.3) 0.536 Females 246 16.8 (16.0–17.6) 182 17.3 (16.3–18.2) 52 15.2 (13.3–17.1) 0.044 Males 110 23.4 (21.7–25.2) 59 24.7 (22.3–27.1) 44 31.4 (28.7–34.2) 0.208 F vs. M P < 0.0001 < 0.0001 < 0.0001 preoperative 24-hour 8-oxoGuo (nmol) all NDH DH DH vs. NDH n mean (95% CI) n mean (95% CI) n mean (95% CI) P All 356 24.4 (23.5–25.3) 241 23.8 (22.8–24.9) 96 26.0 (24.1–27.9) 0.036 Females 246 21.0 (20.2–21.7) 182 20.8 (20.0–21.6) 52 21.4 (19.5–23.3) 0.534 Males 110 32.1 (30.5–33.7) 59 33.1 (31.0–35.2) 44 31.4 (28.7–34.2) 0.324 F vs. M P < 0.0001 < 0.0001 < 0.0001 Data are reported as mean with a 95% confidence interval. NDH, patients with biochemical glucose markers below diagnostic threshold for diabetes and not on antidiabetic treatment; DH, patients with biochemically confirmed diabetes. All patients also include patients for whom we were not able to confirm diabetes status. Exact P-values from unpaired t-tests are reported down to 0.001. https://doi.org/10.1371/journal.pone.0243918.t003 PLOS ONE | https://doi.org/10.1371/journal.pone.0243918 December 14, 2020 8 / 19 PLOS ONE Gastric bypass surgery and urinary markers of DNA/RNA oxidation A table of preoperative excretion levels of 8-oxodG and 8-oxoGuo, normalized to urinary creatinine in the traditional manner, can be found in S2 Table. 24-hour excretion of 8-oxodG and 8-oxoGuo after RYGB After RYGB, a repeated measures ANOVA with group mean substitution of missing postoperative values showed statistically significant changes in the two urinary markers, F (3.748, 1330.535) = 68.014, P < 0.0001 and F (3.601, 1278.454) = 52.668, P < 0.0001 for 8-oxodG and 8-oxoGuo, respectively. Also when missing post-operative values were replaced with the individual pre-operative value, instead of the group mean, results were highly significant (F (3.669, 1302.350) = 20.71, P < 0.0001 for 8-oxodG and F (3.742, 1328.309) = 10.82, P < 0.0001 for 8-oxoGuo). As shown in Fig 2A, a temporary increase was seen for 8-oxodG three months after RYGB, compared to excretion levels before surgery. After the initial increase, levels of 8-oxodG decreased during the rest of the follow-up period, and two years after RYGB, excretion levels were approximately reduced by 12% compared to preoperative levels (Fig 2A). For 8-oxoGuo, we found a similar, although more gradual decrease from three months and onwards without the initially raised level that was seen for 8-oxodG (Fig 2B). The same patterns were seen for patients with and without diabetes (Fig 3A & 3B). It was also seen for female as well as male patients, for smokers as well as non-smokers, and for patients on and not on cholesterol-lowering drugs. In Fig 3A, data from the female subpopulation show that the trend towards lower excretion levels of 8-oxodG in patients with diabetes was consistent throughout the postoperative follow-up period. A similar trend was seen in the smaller male subpopulation. For 8-oxoGuo, postoperative 24-hour excretion levels did not differ between patients with and without diabetes (Fig 3B). The largest difference in excretion levels for 8-oxodG between patients with and without diabetes was seen between patients without diabetes and the group of patients with type 2 diabetes who did not obtain remission in diabetes after RYGB (Fig 3C). This group of patients with diabetes had between 6–7 nmol lower 24-hour excretion level of 8-oxodG, than patients without diabetes. Also, there seemed to be an approximate 4–6 nmol difference between this group and the other group of patients with diabetes (Fig 3C). Between the group of patients who obtained remission in their type 2 diabetes after RYGB and patients without diabetes, no difference was seen. Bodyweight, BMI and markers of glucose and lipid metabolism after RYGB Relative changes in body weight, BMI and markers of glucose metabolism are roughly outlined in Fig 4. As expected after RYGB (and previously described in this study population [33]), there was a rapid loss of bodyweight, with mean BMI dropping approximately ten units during the first six months, in both patients with and without type 2 diabetes. Thereafter, mean weight was stabilized between 88–95 kg. The markers of glucose metabolism (HbA1c, fasting plasma concentrations of glucose, insulin and C-peptide) were improved to a large extent already at three months after the surgery—and HOMA-IR followed. Three months after RYGB, mean HbA1c levels in this sample of the study population had decreased from 39.2 (38.0–40.3) mmol/mol to 34.9 (34.1–35.8) mmol/mol, glucose had decreased from 6.2 (6.0–6.5) mmol/L to 5.7 (5.5–5.8) mmol/L, insulin concentrations were halved from 123 (113–133) pmol/L to 63 (59–67) pmol/L and C-peptide fell from 1234 (1181–1287) pmol/L to 905 (865–946) pmol/L. Mean HOMA-IR fell from 3.0 (2.8–3.1) to a value of 2.1 (2.0–2.2) three months after RYGB. Improvements were also seen for the traditionally measured plasma lipids. Twelve months postoperative there had been a 30% mean increase in HDL-cholesterol and a 25% and 40% decrease in LDL-cholesterol and triglycerides, respectively. PLOS ONE | https://doi.org/10.1371/journal.pone.0243918 December 14, 2020 9 / 19 PLOS ONE Gastric bypass surgery and urinary markers of DNA/RNA oxidation Fig 2. Changes in oxidative stress to DNA and RNA after Roux-en-Y gastric bypass surgery. The figures show 24-hour pre- and postoperative excretion of 8-oxodG (Fig A) and 8-oxoGuo (Fig B) in the whole study population. Data are reported as means with error bars showing the 95% confidence interval of the mean and � symbolizes a significant difference after Bonferroni correction. The number of patients, n, at each time point is shown at the bottom of each figure. https://doi.org/10.1371/journal.pone.0243918.g002 Correlations between 8-oxodG, 8-oxoGuo and other variables Correlation coefficients before and after RYGB, between 8-oxodG, 8-oxoGuo, BMI and markers of glucose- and lipid metabolism, are shown in Table 4. For both 8-oxodG and 8-oxoGuo, there was a strong positive correlation between the traditionally urinary creatinine normalized and the calculated 24-hour excretion levels before as well as after surgery. BMI correlated positively to 8-oxoGuo both before and after surgery, significantly in both patients with and without diabetes, while no correlation was seen between the absolute values of BMI and 8-oxodG. Between delta values, representing the difference between pre- and postoperative BMI and 8-oxodG, respectively, there were, however, a weak but significant negative correlation after RYGB. The largest reductions in both urinary markers were seen in patients with a moderate weight loss, as illustrated in S2 Fig, where individual patient changes in 8-oxodG and 8-oxoGuo 24 months after RYGB are shown in relation to relative postoperative BMI. Although no correlation was found in the entire study population between HbA1c and 8-oxoGuo, there was a weak negative correlation between HbA1c and 8-oxoGuo twelve and twenty-four months after RYGB in patients without diabetes. HbA1c correlated negatively to 8-oxodG both before and after surgery, strongest after RYGB in the group of patients without diabetes. Plasma insulin concentration correlated positively to 8-oxoGuo both before and after RYGB and did not correlate to 8-oxodG. Also, C-peptide and HOMA-IR (but not glucose), showed weak positive correlations to 8-oxoGuo before surgery, as well as twenty-four months after RYGB. C-peptide, HOMA-IR and glucose showed no correlations to 8-oxodG. HDLcholesterol correlated negatively pre- and postoperative to both 8-oxoGuo and 8-oxodG, while no correlation was seen between any of the urinary markers and LDL-cholesterol. Twelve months after RYGB, delta values of HDL-cholesterol and delta values of triglycerides showed negative and positive correlations, respectively, to 8-oxoGuo. PLOS ONE | https://doi.org/10.1371/journal.pone.0243918 December 14, 2020 10 / 19 PLOS ONE Gastric bypass surgery and urinary markers of DNA/RNA oxidation Fig 3. Differences in oxidative stress to DNA and RNA depending on diabetes status. The Figs A & B report data for the female subpopulation divided in two subgroups: patients without diabetes (white bars) and patients with diabetes (black bars). Fig C reports data for the female subpopulation divided in three subgroups: NDH, patients without diabetes (white bars); DH-NDH, patients with diabetes who obtained remission in diabetes after RYGB (dotted bars) and DH-DH, patients with diabetes who did not obtain remission in diabetes after RYGB (black bars). Data are reported as means with error bars showing the 95% confidence interval of the mean and � symbolizes a significant difference after Bonferroni correction. The number of patients, n, at each time point is shown at the bottom of each figure. https://doi.org/10.1371/journal.pone.0243918.g003 Discussion To our knowledge, this study is the first to describe changes in excretion rates of 8-oxodG and 8-oxoGuo, two urinary markers of DNA and RNA oxidation, after RYGB surgery in humans. A main finding was an increase in oxidative stress on DNA the first months after surgery, evidenced by increased excretion of 8-oxodG. In the oxidative stress on RNA, for which 8-oxoGuo excretion serves as a quantitative measure; there was a gradual reduction after surgery and significantly reduced levels twelve months postoperative. Two years after RYGB and a mean weight loss of thirty-five kg and a markedly improved glucose metabolism, including a reduced insulin resistance, excretion rates of both 8-oxodG and 8-oxoGuo had decreased well below preoperative levels. From this, we conclude, that a decrease in oxidative stress on nucleic acids might be contributing to the many long-term benefits of RYGB-surgery in obesity and type 2 diabetes but is PLOS ONE | https://doi.org/10.1371/journal.pone.0243918 December 14, 2020 11 / 19 PLOS ONE Gastric bypass surgery and urinary markers of DNA/RNA oxidation Fig 4. Relative changes in body weight, BMI and markers of glucose metabolism after RYGB. Data are means of levels relative to the preoperative means (in percent) for body weight and BMI (diamonds), C-peptide (black circles), fasting glucose (x’es), HbA1c (triangles), HOMA2-IR (white circles) and Insulin (squares). https://doi.org/10.1371/journal.pone.0243918.g004 less likely a main contributing factor to the immediate effects of RYGB-surgery on weight, glucose- and lipid metabolism within the first postoperative weeks, primarily explained by an increase in insulin sensitivity of the liver [22, 47]. After months and major weight loss also peripheral insulin sensitivity is improved [47]. This is interesting, as a reduced oxidative stress to DNA has been suggested to be one of the acute mechanisms of action of sleeve gastrectomy [14]. We observed a transient increase in 8-oxodG after surgery. Surgery itself is a stressful event with many physiological responses. We cannot decipher specific causes for the increased oxidative stress measures after surgery, but expect that several factors are in play, and that they take some time to normalize after surgery and physiological resetting and reduction of food intake. The reasons why metabolic diseases or the obese condition lead to increased formation of 8oxodG and 8oxoGuo are not known. In isolated obesity without complications, we previously reported that only 8oxoGuo is increased [15], indicating that obesity by itself increases oxidative stress by an unknown mechanism. We suggest that the obese condition is a metabolic stress to the mitochondria and that several physiological responses to obesity and overeating, for instance the release of adipokines, together make the mitochondrial respiratory chain slightly less efficient—not in the context of energy production, but in the context of increased production of ROS such as the superoxide anion, hydrogen peroxide and the hydroxy radical, that in turn will oxidize the RNAs in the vicinity of the mitochondria. RNA will in this situation function as a “photographic plate” for mitochondrial ROS production. Also, oxidized PLOS ONE | https://doi.org/10.1371/journal.pone.0243918 December 14, 2020 12 / 19 PLOS ONE Gastric bypass surgery and urinary markers of DNA/RNA oxidation Table 4. Correlations between 8-oxodG, 8-oxoGuo, BMI and markers of glucose and lipid metabolism. 24-hour 8-oxodG (nmol) before surgery 24-hour 8-oxoGuo 8-oxodG (nmol/mmol creatinine) 8-oxoGuo (nmol/mmol creatinine) BMI, All BMI, NDH BMI, DH HbA1c, All HbA1c, NDH HbA1c, DH Insulin Glucose C-peptide HOMA-IR Total Cholesterol HDL-Cholesterol 12 months after RYGB 24 months after RYGB rs 0.643�� 0.692�� 0.645�� P < 0.0001 < 0.0001 < 0.0001 n 356 228 168 rs 0.755�� 0.849�� 0.837�� P < 0.0001 < 0.0001 < 0.0001 n 356 228 168 24-hour 8-oxoGuo (nmol) Δ:Δ 12 months after RYGB rs before surgery 12 months after RYGB 24 months after RYGB 0.578�� 0.745�� 0.769�� P < 0.0001 < 0.0001 < 0.0001 n 356 229 168 Δ:Δ 12 months after RYGB rs 0.032 0.075 0.047 -0.189�� 0.137�� 0.207�� 0.235�� -0.057 P 0.553 0.262 0.546 0.004 0.009 0.002 0.002 0.391 n 356 228 168 228 356 229 168 229 rs 0.063 0.128 0.087 -0.159 0.189�� 0.244�� 0.144 -0.071 P 0.330 0.118 0.366 0.051 0.003 0.002 0.131 0.389 n 241 150 111 150 241 151 111 151 � �� rs 0.033 0.068 0.092 -0.220 0.217 0.234 0.426 0.004 P 0.753 0.590 0.538 0.078 0.034 0.061 0.003 0.972 n 96 65 47 65 96 65 47 65 rs -0,112� -0.282�� -0.319�� -0.68 0.086 -0.093 -0.003 -0.035 P < 0.037 < 0.0001 < 0.0001 0.317 0.109 0.166 0.965 0.601 n 348 225 164 219 348 226 164 220 rs -0.103 -0.270�� -0.414�� -0.119 0.031 -0.188� -0.241� -0.061 P 0.115 0.001 < 0.0001 0.155 0.637 0.021 0.012 0.463 n 235 148 109 144 235 149 109 145 rs -0.031 -0.245 -0.297� -0.166 -0.061 -0.069 0.063 -0.112 P 0.770 0.051 0.048 0.196 0.562 0.590 0.681 0.385 n 94 64 45 62 94 64 45 62 rs 0.044 0.051 0.034 -0.104 0.193�� 0.155� 0.208� -0.075 P 0.416 0.457 0.677 0.139 < 0.001 0.023 0.01 0.283 n 346 213 152 205 346 214 152 206 rs -0.088 -0.128 -0.131 -0.116 0.062 0.055 0.072 -0.070 P 0.098 0.057 0.095 0.813 0.248 0.416 0.361 0.302 n 351 223 164 220 351 224 164 221 rs 0.020 -0.040 0.017 -0.064 0.170�� 0.089 0.186� -0.011 P 0.709 0.549 0.831 0.345 0.001 0.183 0.017 0.875 221 n 349 225 164 220 349 226 164 rs -0.008 -0.059 -0.002 -0.043 0.158�� 0.101 0.203�� -0.012 P 0.879 0.380 0.977 0.542 0.004 0.134 0.009 0.868 n 336 221 162 207 336 222 162 208 � rs 0.034 -0.037 -0.008 0.010 -0.133 P 0.527 0.582 0.907 0.846 0.045 0.158 n 354 227 226 354 228 227 rs -0.150�� -0.188�� -0.111 -.193�� -0.145� -0.181�� 0.094 P 0.005 0.004 0.097 < 0.001 0.029 0.006 n 354 227 226 354 228 227 (Continued ) PLOS ONE | https://doi.org/10.1371/journal.pone.0243918 December 14, 2020 13 / 19 PLOS ONE Gastric bypass surgery and urinary markers of DNA/RNA oxidation Table 4. (Continued) 24-hour 8-oxodG (nmol) before surgery LDL-Cholesterol Triglycerides 12 months after RYGB 24 months after RYGB 24-hour 8-oxoGuo (nmol) Δ:Δ 12 months after RYGB before surgery 12 months after RYGB 24 months after RYGB Δ:Δ 12 months after RYGB rs 0.103 0.055 0.017 0.023 -0.072 0.112 P 0.054 0.405 0.798 0.664 0.281 0.097 n 349 227 221 349 228 222 rs 0.002 0.061 0.040 0.133� 0.060 0.205�� P 0.971 0.357 0.547 0.012 0.366 0.002 n 354 227 226 354 228 227 Exact P-values are reported down to 0.001. Below 0.001, P-values are reported as <0.001 or <0.0001. � and �� indicates significance on the 0.05- and 0.01-level, respectively. For BMI and HbA1c, correlations are shown for the whole study population and for the subpopulations of patients with (DH) and without diabetes (NDH). For BMI, HOMA-IR and lipids, correlations between the delta values 12 months after Roux-en-Y gastric bypass (RYGB) are shown in the columns marked Δ:Δ for 8-oxodG and 8-oxoGuo, respectively. https://doi.org/10.1371/journal.pone.0243918.t004 RNAs have been shown to have aberrant functions [7] with implications for disease processes [5]. A reduction in bodyweight has been reported to reduce other markers of oxidative stress [48, 49], but so far, only few interventions except for smoking cessation, olive oil intake [46] and perhaps sleeve gastrectomy [14] have been able to reduce levels of urinary markers of DNA or RNA oxidation. Nevertheless, since ROS clearly play a role in pathogenesis of various diseases, (focus has been on the toxicological aspects of high levels of oxidative stress, however it is now realized that modifications in the redox balance is a multifaceted regulatory mechanism in normal physiological situations [5]), preventive and therapeutic strategies that aim to restore redox homeostasis may be an intuitive approach with a large potential to protect against metabolic diseases [3–5]. The present study showed that RYGB-induced weight loss does indeed reduce oxidative stress on DNA and RNA. The changing physiology following RYGB might therefore provide a way to understand the mechanisms of action that improve redox-homeostasis. In patients with persisting hyperglycemia after RYGB, excretion rates of 8-oxodG were lower after surgery than for patients without type 2 diabetes, independently of gender and smoking status. It is not clear, whether the reason for this difference is a smaller increase in oxidant formation after RYGB in the group of patients who did not respond to the surgery with diabetes remission and therefore continued diabetes treatment; if the proportion of antioxidant defense mechanisms, for example dietary or enzymatic anti-oxidants, might be higher in this group; or if DNA’s availability for oxidation by some reason might be lower. A higher HbA1c in the group of patients without diabetes was also associated to lower levels of 8-oxodG after RYGB, suggesting that changes in 8-oxodG after RYGB somehow are modified by glucose metabolism. An association between 8-oxoGuo and diabetes has been described in more than one population [16, 50]. These studies studied excretion levels of 8-oxoGuo, normalized to urinary creatinine. In our obese study population, we could not confirm the presence of a similar association after adjusting for differences in weight, height and kidney function—the variables that were included in the physiological model for estimating 24-hour excretion. Although there was no convincing association between 24-hour 8-oxoGuo excretion and HbA1c, we did find higher 8-oxoGuo excretion levels with increasing insulin, c-peptide and HOMA-IR, PLOS ONE | https://doi.org/10.1371/journal.pone.0243918 December 14, 2020 14 / 19 PLOS ONE Gastric bypass surgery and urinary markers of DNA/RNA oxidation indicating a connection between 8-oxoGuo and the hyper-insulinemic, insulin resistant state of metabolic disease, which in this morbidly obese study population was not exclusive for patients with diabetes. These findings also suggest that bodyweight, height and perhaps kidney function should be taken into consideration and corrected for, when urinary levels of oxidative damage to nucleic acids are compared between study populations. This appears rational, as the size and composition of the body have impact on the metabolic rate that affect the production of ROS in the mitochondria [51] and that there is a direct association between ROS-formation and oxidative damage to DNA and RNA [5]. Although the exact source of ROS that oxidize DNA is not known in detail, it is believed, partly to be overlapping and partly, to some extent also to differ from the source of ROS that oxidize RNA. Although RNA and DNA oxidation are correlated, their prognostic values differ in type 2 diabetes, consistent with the view that they represent different mechanisms. The exact mechanisms need to be explored in future studies. Between female and male subjects, large differences in excretion levels of both urinary markers were observed in this study. Although gender for long has been recognized as a confounding factor for 8-oxodG [45], details of the physiological background of these differences are not known. It is also mainly unknown, and merit further studies, if the gender differences in levels of 8-oxodG and 8-oxoGuo are of importance for health outcomes. In summary, this study confirms previously documented associations between 8-oxoGuo and obesity, but questions previously documented associations between 8-oxoGuo and diabetes and HbA1c, respectively, pointing more towards a connection to hyperinsulinemia and insulin-resistance. We conclude that oxidative damage to nucleic acids, reflected in the excretion of 8-oxodG and 8-oxoGuo, gradually decreased postoperatively (except for a temporary increase in 8-oxodG immediately after surgery) and had decreased significantly two years after RYGB. This indicates that a reduction in oxidative stress could contribute to the many longterm benefits of RYGB-surgery in obesity and type 2 diabetes, including improved longevity. Supporting information S1 Fig. Research population inclusion and exclusion criteria. (TIF) S2 Fig. Individual changes in BMI and urinary markers 24 months after RYGB. The graph is a x-y plot, where delta-values of 8-oxodG (A) and 8-oxoGuo (B) are plotted against the relative BMI for individual patients, 24 months after RYGB. On the x-axis, 0 nmol represents no change in urinary excretion of the marker. On the y-axis, 100% represents the preoperative BMI-value. (TIF) S1 Table. Preoperative clinical and laboratory data for all Roux-en-Y Gastric Bypass-operated patients and patients divided in subgroups according to diabetes status. Data are reported as Mean (SD), except for the parameters reported as a percentage or number. Age is on the day of Roux-en-Y gastric bypass (RYGB) surgery. Clinical data represent the closest available before surgery. NDH, patients with biochemical glucose markers below diagnostic threshold for diabetes and not on antidiabetic treatment; DH, patients with biochemically confirmed diabetes; DH-NDH, patients who obtained remission of diabetes after RYGB; DH-DH, patients who did not obtain remission in diabetes after RYGB; SU, Sulfonylurea; GLP-1, Glucagon-like peptide-1 analogue; DPP4, Dipeptidyl peptidase-4 inhibitor. � All patients also include patients for whom we were not able to confirm diabetes status. †All patients with diabetes also include patients without hyperglycemia after RYGB, but who continued antidiabetic PLOS ONE | https://doi.org/10.1371/journal.pone.0243918 December 14, 2020 15 / 19 PLOS ONE Gastric bypass surgery and urinary markers of DNA/RNA oxidation medicine. ‡Apart from the HOMA-IR score and eGFR, biochemical variables are plasma or blood concentrations, unless stated otherwise with U, for urinary. Insulin, C-peptide and Glucose are fasting values. Exact p-values from the one-way ANOVA, followed by a Tukey post hoc test, are reported down to 0.001. Below 0.001, P-values are reported as < 0.001 or < 0.0001. A Welch—Satterthwaite correction followed by a Games—Howell post hoc test have been used as appropriate, to adjust for unequal variances and for HbA1c, glucose and triglycerides, significant differences were confirmed with a Kruskal-Wallis H-test, as these variables did not have a normal distribution. (DOCX) S2 Table. Preoperative 8-oxodG and 8-oxoGuo, normalized to urinary creatinine. Data are reported as mean with a 95% confidence interval. NDH, patients with biochemical glucose markers below diagnostic threshold for diabetes and not on antidiabetic treatment; DH, patients with biochemically confirmed diabetes. All patients also include patients for whom we were not able to confirm diabetes status. Exact P-values from unpaired t-tests are reported down to 0.001. (DOCX) Acknowledgments We would like to thank Jette Nymann and Bente Elmfeldt Madsen for assistance with biobank sample handling and analysis of cystatin C, and Katja Luntang Christensen for analysis of 8oxoGuo, 8oxodG and urinary creatinine. Author Contributions Conceptualization: Mogens Fenger, Sten Madsbad, Henrik Enghusen Poulsen. Data curation: Elin Rebecka Carlsson. Formal analysis: Elin Rebecka Carlsson. Funding acquisition: Sten Madsbad. Investigation: Elin Rebecka Carlsson. Methodology: Elin Rebecka Carlsson, Mogens Fenger, Henrik Enghusen Poulsen. Project administration: Elin Rebecka Carlsson, Mogens Fenger, Henrik Enghusen Poulsen. Resources: Trine Henriksen, Dorte Worm, Dorte Lindqvist Hansen, Sten Madsbad, Henrik Enghusen Poulsen. Supervision: Mogens Fenger, Laura Kofoed Kjaer, Sten Madsbad, Henrik Enghusen Poulsen. Validation: Trine Henriksen, Henrik Enghusen Poulsen. Writing – original draft: Elin Rebecka Carlsson. Writing – review & editing: Elin Rebecka Carlsson, Mogens Fenger, Trine Henriksen, Laura Kofoed Kjaer, Dorte Worm, Dorte Lindqvist Hansen, Sten Madsbad, Henrik Enghusen Poulsen. References 1. Hruby A, Manson JAE, Qi L, Malik VS, Rimm EB, Sun Q, et al. Determinants and consequences of obesity. Am J Public Health. 2016; 106(9):1656–62. https://doi.org/10.2105/AJPH.2016.303326 PMID: 27459460 PLOS ONE | https://doi.org/10.1371/journal.pone.0243918 December 14, 2020 16 / 19 PLOS ONE Gastric bypass surgery and urinary markers of DNA/RNA oxidation 2. Marseglia L, Manti S, D’Angelo G, Nicotera A, Parisi E, Di Rosa G, et al. Oxidative stress in obesity: A critical component in human diseases. Int J Mol Sci. 2015; 16(1):378–400. 3. Rani V, Deep G, Singh RK, Palle K, Yadav UCS. Oxidative stress and metabolic disorders: Pathogenesis and therapeutic strategies. Life Sci [Internet]. 2016; 148:183–93. Available from: http://dx.doi.org/ 10.1016/j.lfs.2016.02.002 PMID: 26851532 4. 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www.nature.com/scientificreports OPEN Author Correction: A framework for in situ molecular characterization of coral holobionts using nanopore sequencing Quentin Carradec, Julie Poulain, Emilie Boissin, Benjamin C. C. Hume, Christian R. Voolstra, Maren Ziegler, Stefan Engelen, Corinne Cruaud, Serge Planes & Patrick Wincker Correction to: Scientific Reports https​://doi.org/10.1038/s4159​8-020-72589​-0, published online 27 May 2020 The Acknowledgements section in this Article is incomplete. “This project has been funded through the Tara Pacific consortium, France Genomique Grant Number ANR-10INBS-09, and the Genoscope/CEA. We are keen to thank the commitment of the people and the following institutions and sponsors who made this singular expedition possible: CNRS, CSM, PSL, KAUST, Genoscope/CEA, ANR-CORALGENE, agnès b., the Veolia Environment Foundation, Region Bretagne, Serge Ferrari, Billerudkorsnas, AmerisourceBergen Company, Lorient Agglomération, Oceans by Disney, the Prince Albert II de Monaco Foundation, L′Oreal, Biotherm, France Collectivites, Kankyo Station, Fonds Francais pour l′Environnement Mondial (FFEM), Etienne Bourgois, UNESCO-IOC, the Tara Foundation teams and crew. Tara Pacific would not exist without the continuous support of the participating institutes. This publication is number 5 of the Tara Pacific Consortium.” should read: “This project has been funded through the Tara Pacific consortium, France Genomique Grant Number ANR-10INBS-09, and the Genoscope/CEA. We are keen to thank the commitment of the people and the following institutions and sponsors who made this singular expedition possible: CNRS, CSM, PSL, KAUST, Genoscope/CEA, ANR-CORALGENE, agnès b., the Veolia Environment Foundation, Region Bretagne, Serge Ferrari, Billerudkorsnas, AmerisourceBergen Company, Lorient Agglomération, Oceans by Disney, the Prince Albert II de Monaco Foundation, L′Oreal, Biotherm, France Collectivites, Kankyo Station, Fonds Francais pour l′Environnement Mondial (FFEM), Etienne Bourgois, UNESCO-IOC, the Tara Foundation teams and crew. Tara Pacific would not exist without the continuous support of the participating institutes. We also thank Professor Russel Clark Perembo, Alfred Yohang Ko’ou, Augustine Mungkaje and Professor Simon Saulei, researchers of the Marine Scientific Research Committee and the National Research Institute of Papua New Guinea for the authorization of coral sampling and their valuable assistance during Tara expedition in Papua New Guinea. This publication is number 5 of the Tara Pacific Consortium.” Additionally, the information about research permissions is not included in the Article. The sampling and research described in this paper was approved by the Marine Scientific Research committee and the department of foreign affairs and trade of the independent state of Papua New Guinea, under the diplomatic clearance number 0232. Scientific Reports | (2021) 11:2076 | https://doi.org/10.1038/s41598-021-81544-6 1 Vol.:(0123456789) www.nature.com/scientificreports/ 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://creat​iveco​mmons​.org/licen​ses/by/4.0/. © The Author(s) 2021 Scientific Reports | Vol:.(1234567890) (2021) 11:2076 | https://doi.org/10.1038/s41598-021-81544-6 2 

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Delving into the Motivations behind Tech Adoption in Entrepreneurship Yuyeong Park New York University Research Article Keywords: Technology adoption, entrepreneurship, motivations, economic incentives, strategic imperatives, personal aspirations, qualitative research Posted Date: March 18th, 2024 DOI: https://doi.org/10.21203/rs.3.rs-4114816/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: The authors declare no competing interests. Page 1/13 Abstract This qualitative study delves into the intricate motivations driving technology adoption within entrepreneurial endeavors. By employing in-depth interviews and thematic analysis, it uncovers the diverse array of factors shaping entrepreneurs' decisions to integrate technology into their ventures. Economic incentives, strategic imperatives, and personal aspirations emerge as key drivers behind technology adoption. Economic motivations encompass the desire to enhance operational efficiency, reduce costs, and gain a competitive edge. Strategic imperatives drive entrepreneurs to leverage technology for long-term growth, scalability, and market penetration. Personal motivations, including a passion for innovation, autonomy, and societal impact, also play a significant role in shaping technology adoption decisions. The study reveals a dynamic and iterative technology adoption process characterized by experimentation, learning, and adaptation. Despite challenges such as technical complexity, resource constraints, and resistance to change, entrepreneurs report positive outcomes from technology adoption, including improved efficiency, increased productivity, and enhanced competitiveness. Moreover, technology adoption has broader implications for innovation, job creation, and societal impact. These findings contribute to a deeper understanding of the dynamics between technology adoption and entrepreneurship. The study offers actionable insights for stakeholders, including policymakers, practitioners, and researchers, seeking to support and promote technology-enabled entrepreneurship. By aligning technological investments with entrepreneurial goals and values, fostering a supportive ecosystem for technology adoption, and promoting digital literacy and access to resources, stakeholders can harness the transformative potential of technology to drive innovation, growth, and positive change in entrepreneurship and society. 1. Introduction The rapid advancement of technology has profoundly transformed the entrepreneurial landscape, shaping the way businesses operate, compete, and innovate. In recent years, the integration of technology into entrepreneurial ventures has become increasingly ubiquitous, with entrepreneurs leveraging a myriad of digital tools, platforms, and solutions to drive growth, enhance efficiency, and seize opportunities in the marketplace (Mollick, 2021). From artificial intelligence and blockchain to cloud computing and the Internet of Things (IoT), the spectrum of available technologies offers entrepreneurs unprecedented opportunities to disrupt industries, create value, and address complex challenges (Kohli & Melville, 2020). The motivations behind technology adoption in entrepreneurship constitute a critical area of inquiry, as they underpin the decisions and actions of entrepreneurs in embracing and deploying technological innovations within their ventures. Understanding these motivations is essential for elucidating the dynamics of technology adoption processes, as well as for informing strategies aimed at fostering innovation and entrepreneurship in today's digital era. While prior research has examined various aspects of technology adoption in entrepreneurship, there remains a need for a comprehensive exploration of the underlying motivational forces that drive entrepreneurs towards embracing technology in their ventures. This qualitative research endeavors to address this gap by delving into the motivations Page 2/13 behind technology adoption in entrepreneurship, elucidating the diverse array of factors that influence entrepreneurs' decisions to integrate technology into their business operations. By employing in-depth interviews and thematic analysis, this study seeks to uncover the complex interplay of economic, strategic, and personal motivations that drive entrepreneurs to adopt technology, thereby contributing to a deeper understanding of the mechanisms and implications of technology adoption in entrepreneurial endeavors. Technology adoption in entrepreneurship encompasses a broad spectrum of activities, ranging from the adoption of software applications for internal operations to the development of cuttingedge products or services enabled by emerging technologies. At its core, technology adoption involves the deliberate and systematic incorporation of technological innovations into business processes, products, or services, with the aim of achieving specific objectives or gaining competitive advantages (Venkatesh et al., 2020). This process is inherently dynamic and context-dependent, shaped by a myriad of internal and external factors that influence entrepreneurs' perceptions, attitudes, and behaviors towards technology adoption (Lyytinen & Damsgaard, 2021). One of the key drivers of technology adoption in entrepreneurship is the pursuit of economic benefits, whereby entrepreneurs seek to leverage technology as a means of enhancing operational efficiency, reducing costs, and increasing profitability (Pavlou & El Sawy, 2020). In today's hypercompetitive business environment, where margins are often thin and market dynamics are constantly evolving, the adoption of technology offers entrepreneurs a means of optimizing resource allocation, streamlining processes, and maximizing productivity (Mithas et al., 2022). By automating repetitive tasks, improving decision-making processes, and enabling real-time data analytics, technology empowers entrepreneurs to allocate their time and resources more effectively, thereby enabling them to focus on strategic priorities and value-creating activities (Bapna et al., 2018). Furthermore, the adoption of technology in entrepreneurship is driven by strategic imperatives, whereby entrepreneurs seek to leverage technology as a tool for achieving long-term growth, scalability, and market competitiveness (Zott et al., 2021). In an increasingly interconnected and digitized marketplace, technology plays a pivotal role in enabling entrepreneurs to reach new markets, expand their customer base, and capitalize on emerging opportunities (Davenport et al., 2019). By embracing digital platforms, e-commerce solutions, and data-driven marketing strategies, entrepreneurs can overcome geographical barriers, target niche segments, and create personalized experiences for their customers, thereby gaining a competitive edge in the marketplace (Gupta et al., 2021). Moreover, technology adoption in entrepreneurship is driven by personal motivations, encompassing entrepreneurs' intrinsic desires, values, and aspirations (Shane & Venkataraman, 2020). For many entrepreneurs, the pursuit of innovation and creativity serves as a primary motivator for embracing technology, as they seek to develop novel solutions, disrupt existing markets, and push the boundaries of what is possible (Foss et al., 2019). The intrinsic satisfaction derived from creating something new, making a positive impact, or solving a pressing problem can serve as a powerful catalyst for technology adoption, driving entrepreneurs to overcome barriers and pursue ambitious goals (George et al., 2021). Additionally, personal autonomy and freedom are important motivators for entrepreneurs, as they seek to escape the constraints of traditional employment and pursue their passions, interests, and visions (Baum et al., 2020). By leveraging technology to launch and grow their ventures, entrepreneurs can exercise greater control over their destinies, make autonomous decisions, and shape their organizations in alignment with their values and Page 3/13 objectives (Sine et al., 2019). The flexibility and agility afforded by technology enable entrepreneurs to adapt to changing market conditions, experiment with new ideas, and pivot their strategies in response to feedback and learning (Thompson et al., 2022). Furthermore, many entrepreneurs are motivated by a desire to make a positive impact on society and the environment, leveraging technology as a means of addressing pressing social, environmental, and economic challenges (Wry et al., 2021). Whether through the development of sustainable technologies, the provision of essential services to underserved communities, or the promotion of social entrepreneurship initiatives, technology-enabled ventures have the potential to generate meaningful and lasting impact on society (Battilana et al., 2017). By aligning their ventures with social or environmental causes, entrepreneurs can attract support from stakeholders, differentiate their offerings, and create shared value for both business and society (Dacin et al., 2019). 2. Literature Review The integration of technology into entrepreneurial ventures has garnered significant attention from scholars, practitioners, and policymakers alike, reflecting the growing recognition of technology as a key driver of innovation, growth, and competitiveness in today's economy. The literature on technology adoption in entrepreneurship spans multiple disciplines, encompassing fields such as entrepreneurship, innovation management, information systems, and strategic management. This section provides an overview of key themes, theories, and empirical findings from recent studies on technology adoption in entrepreneurship, with a focus on understanding the motivations, processes, and outcomes of technology adoption among entrepreneurs. 2.1 Motivations for Technology Adoption in Entrepreneurship A central focus of research on technology adoption in entrepreneurship is the examination of the motivations that drive entrepreneurs towards embracing technology in their ventures. Scholars have identified a diverse array of economic, strategic, and personal motivations that influence entrepreneurs' decisions and behaviors in adopting technology (Hasan Emon et al., 2023). Economic motivations, such as the desire to enhance efficiency, reduce costs, and increase profitability, are commonly cited as primary drivers of technology adoption among entrepreneurs (Mithas et al., 2022). By leveraging technology to automate routine tasks, optimize resource allocation, and improve decision-making processes, entrepreneurs can achieve operational efficiencies and gain a competitive edge in the marketplace (Pavlou & El Sawy, 2020). Moreover, strategic motivations play a significant role in shaping entrepreneurs' decisions to adopt technology, as they seek to leverage technology as a tool for achieving long-term growth, scalability, and market competitiveness (Zott et al., 2021). Technology enables entrepreneurs to expand their reach, penetrate new markets, and capitalize on emerging opportunities, thereby enhancing their prospects for sustainable growth and success (Davenport et al., 2019). By embracing digital platforms, e-commerce solutions, and data-driven strategies, entrepreneurs can differentiate their offerings, target niche segments, and create value for customers, thereby strengthening their competitive Page 4/13 position in the marketplace (Kohli & Melville, 2020). Furthermore, personal motivations play a crucial role in driving technology adoption among entrepreneurs, reflecting their intrinsic desires, values, and aspirations (Shane & Venkataraman, 2020). Many entrepreneurs are driven by a passion for innovation and creativity, seeking to develop novel solutions, disrupt existing markets, and make a positive impact on society (Foss et al., 2019). The autonomy and freedom afforded by entrepreneurship also serve as important motivators, as entrepreneurs seek to escape the constraints of traditional employment and pursue their own visions, interests, and goals (M. H. Emon & Nipa, 2024). Additionally, the desire to create social or environmental impact motivates entrepreneurs to leverage technology as a means of addressing pressing societal challenges and advancing the greater good (Battilana et al., 2017). 2.2 Processes of Technology Adoption in Entrepreneurship The process of technology adoption in entrepreneurship is characterized by a series of stages, decisions, and actions that entrepreneurs undertake as they integrate technology into their ventures. Drawing on insights from diffusion of innovations theory (M. M. H. Emon & Khan, 2023), scholars have identified several key stages in the technology adoption process, including awareness, interest, evaluation, trial, and adoption (Venkatesh et al., 2020). At each stage, entrepreneurs must navigate a complex array of factors, including technological characteristics, organizational context, market dynamics, and personal preferences, which shape their perceptions, attitudes, and intentions towards technology adoption (Lyytinen & Damsgaard, 2021). The initial stage of the technology adoption process involves creating awareness and generating interest among entrepreneurs about the potential benefits and applications of technology in their ventures (Mollick, 2021). Entrepreneurs may become aware of new technologies through various channels, such as industry events, trade publications, social networks, or personal experiences, which spark their curiosity and interest in exploring further (Mithas et al., 2022). As entrepreneurs delve deeper into the potential benefits and implications of technology adoption, they engage in a process of evaluation, weighing the costs, benefits, risks, and uncertainties associated with adopting and implementing technology in their ventures (Pavlou & El Sawy, 2020). The trial stage involves experimenting with technology on a small scale, testing its feasibility, functionality, and compatibility within the organizational context (Kohli & Melville, 2020). Entrepreneurs may pilot new technologies in specific areas of their business, gather feedback from users, and iterate on their implementation strategies based on real-world experiences and observations (Davenport et al., 2019). Through this iterative process of trial and error, entrepreneurs learn about the capabilities and limitations of technology, as well as its potential to create value and drive performance improvements in their ventures (Zott et al., 2021). The final stage of the technology adoption process entails the full-scale adoption and integration of technology into the core operations, products, or services of the venture (Shane & Venkataraman, 2020). This involves making strategic investments in technology infrastructure, human capital, and organizational capabilities, as well as aligning technology adoption efforts with broader business objectives and strategies (Foss et al., 2019). Successful technology adoption requires entrepreneurs to overcome barriers and challenges, such as resistance to change, resource constraints, technical complexities, and cultural factors, by fostering a supportive organizational culture, building internal capabilities, and leveraging external resources and partnerships (Baum et al., 2020). Page 5/13 2.3 Outcomes of Technology Adoption in Entrepreneurship The outcomes of technology adoption in entrepreneurship encompass a range of tangible and intangible benefits, which accrue to entrepreneurs, their ventures, and the broader ecosystem. Scholars have identified various dimensions of outcomes, including economic performance, innovation capabilities, competitive advantage, and societal impact (Battilana et al., 2017). From an economic perspective, technology adoption can lead to improvements in productivity, efficiency, and profitability, as entrepreneurs leverage technology to optimize processes, reduce costs, and increase revenues (Gupta et al., 2021). Moreover, technology adoption enables entrepreneurs to access new markets, reach new customers, and scale their ventures more rapidly, thereby enhancing their prospects for growth and expansion (Mithas et al., 2022). Furthermore, technology adoption enhances entrepreneurs' innovation capabilities, as they leverage technology to develop new products, services, and business models that create value for customers and differentiate their offerings in the marketplace (Pavlou & El Sawy, 2020). By embracing emerging technologies such as artificial intelligence, machine learning, and blockchain, entrepreneurs can unlock new sources of value creation, drive product innovation, and transform industries (Kohli & Melville, 2020). Additionally, technology adoption enables entrepreneurs to adapt to changing market conditions, anticipate future trends, and capitalize on emerging opportunities, thereby enhancing their agility and resilience in a dynamic business environment (Davenport et al., 2019). Moreover, technology adoption can create competitive advantage for entrepreneurs by enabling them to leverage digital capabilities, data-driven insights, and network effects to outperform rivals and capture market share (Zott et al., 2021). Entrepreneurs who are early adopters of technology can establish themselves as industry leaders, disrupt incumbents, and shape the direction of their industries through innovation and strategic differentiation (Shane & Venkataraman, 2020). Additionally, technology adoption can enhance entrepreneurs' ability to attract and retain talent, as employees are drawn to innovative and forward-thinking organizations that offer opportunities for learning, growth, and meaningful work (Foss et al., 2019). Furthermore, technology adoption in entrepreneurship has the potential to generate positive societal impact by addressing pressing social, environmental, and economic challenges (Baum et al., 2020). Entrepreneurs who develop technology-enabled solutions to issues such as healthcare access, education inequality, environmental sustainability, and economic development can create value for society while also building successful and sustainable ventures (Battilana et al., 2017). Moreover, technology adoption can empower marginalized communities, enable inclusive economic growth, and foster social innovation by democratizing access to resources, opportunities, and information (Gupta et al., 2021). 3. Research Methodology The research methodology employed in this study was designed to explore the motivations behind technology adoption in entrepreneurship through in-depth interviews and thematic analysis. A qualitative approach was chosen to enable a rich exploration of the diverse array of factors influencing entrepreneurs' decisions to integrate technology into their ventures. Participants were selected using Page 6/13 purposive sampling, with the aim of capturing a diverse range of perspectives and experiences related to technology adoption in entrepreneurship. Potential participants were identified through professional networks, industry associations, and online communities, and were invited to participate in the study based on their involvement in technology-enabled ventures or their expertise in entrepreneurship and technology adoption. Semi-structured interviews were conducted with each participant to gather qualitative data on their motivations, perceptions, and experiences related to technology adoption in their ventures. The interview protocol was designed to elicit detailed narratives and insights from participants, covering topics such as their reasons for adopting technology, the challenges they encountered, and the outcomes they experienced as a result of technology adoption. A total of 20 interviews were conducted with entrepreneurs from a variety of industries, including technology startups, e-commerce businesses, and social enterprises. The interviews were conducted one-on-one, either in person or via video conferencing, and ranged in duration from 45 minutes to 90 minutes. All interviews were audio-recorded with the consent of the participants and transcribed verbatim for analysis. Thematic analysis was employed to identify recurring patterns, themes, and insights across the interview data. The analysis process involved several stages, including familiarization with the data, coding of the transcripts, identification of themes, and interpretation of the findings (Braun & Clarke, 2019). Codes were initially generated inductively based on the interview data, and were subsequently organized into broader themes through iterative rounds of coding and discussion. Trustworthiness and rigor were ensured through several strategies, including member checking, peer debriefing, and triangulation of data sources (Creswell & Creswell, 2017). Member checking involved sharing preliminary findings with participants to validate the accuracy and interpretation of their responses. Peer debriefing involved seeking input from colleagues and experts in qualitative research to ensure the credibility and trustworthiness of the analysis. Triangulation of data sources involved comparing and contrasting findings from different interviews to identify convergent and divergent perspectives on the research topic. Ethical considerations were paramount throughout the research process, with measures taken to protect the confidentiality and anonymity of participants, obtain informed consent, and ensure that the research was conducted in accordance with ethical guidelines and principles (Bryman, 2016). Participants were provided with information about the study objectives, procedures, and potential risks, and were given the opportunity to withdraw from the study at any time without consequences. 4. Results and Findings The results and findings of the study provide insights into the motivations behind technology adoption in entrepreneurship, as gleaned from in-depth interviews with 20 entrepreneurs. Thematic analysis of the interview data revealed a rich tapestry of factors influencing entrepreneurs' decisions to integrate technology into their ventures, encompassing economic, strategic, and personal motivations. 4.1 Economic Motivations Economic considerations emerged as a primary driver of technology adoption among entrepreneurs. Many participants cited the desire to enhance operational efficiency, reduce costs, and increase Page 7/13 profitability as key motivations for adopting technology in their ventures. For example, one participant, a founder of a software startup, noted, "We adopted technology to streamline our processes and improve efficiency. By automating repetitive tasks and leveraging data analytics, we were able to reduce our operating costs and increase our bottom line." Similarly, another participant, the owner of an e-commerce business, highlighted the role of technology in driving cost savings and improving business performance. "Technology has allowed us to optimize our supply chain, manage inventory more effectively, and improve the customer experience," they remarked. "As a result, we've been able to lower our costs and increase our profitability, which has been crucial for our growth and sustainability." 4.2 Strategic Motivations Strategic imperatives also played a significant role in driving technology adoption among entrepreneurs. Participants emphasized the importance of leveraging technology as a tool for achieving long-term growth, scalability, and market competitiveness. For instance, one participant, the CEO of a digital marketing agency, discussed how technology had enabled them to expand their client base and penetrate new markets. "By harnessing the power of digital platforms and data-driven marketing strategies, we've been able to reach customers across the globe and scale our business rapidly," they explained. Similarly, another participant, the co-founder of a fintech startup, highlighted the strategic importance of technology in disrupting traditional financial services and capturing market share. "Our goal was to democratize access to financial services and empower underserved communities through technology," they noted. "By leveraging blockchain and mobile technology, we've been able to offer innovative products and services that address the needs of our target market, positioning us as a leader in the industry." 4.3 Personal Motivations Personal motivations also emerged as influential factors driving technology adoption among entrepreneurs. Many participants expressed a passion for innovation, a desire for autonomy, and a commitment to making a positive impact on society as key drivers of their decision to adopt technology in their ventures. For example, one participant, a social entrepreneur, discussed how their personal values had guided their technology adoption efforts. "Our venture is driven by a mission to create social change and improve the lives of others," they stated. "Technology has been instrumental in helping us achieve our goals, whether it's through developing new solutions to address social problems or leveraging digital platforms to amplify our impact." Similarly, another participant, the founder of a healthcare startup, emphasized the role of personal passion and motivation in driving their technology adoption journey. "I've always been passionate about using technology to improve healthcare outcomes and enhance patient care," they explained. "That passion has been the driving force behind our efforts to develop innovative medical devices and digital health solutions that have the potential to transform the healthcare industry." 4.4 Barriers and Challenges Despite the many benefits of technology adoption, participants also identified several barriers and challenges that they encountered along the way. Technical complexity, resource constraints, and Page 8/13 resistance to change were commonly cited as obstacles to technology adoption in entrepreneurship. For example, one participant discussed the challenges of integrating new technologies into their existing systems and workflows. "There's often a steep learning curve associated with adopting new technologies, especially for small businesses with limited resources," they noted. "We had to invest time and effort into training our team and overcoming resistance to change in order to successfully implement new technologies." Similarly, another participant highlighted the financial constraints faced by startups and small businesses in adopting technology. "Cost is always a concern when it comes to technology adoption," they remarked. "Investing in new software, hardware, and infrastructure can be prohibitively expensive, especially for cash-strapped startups with limited funding." Moreover, organizational culture and resistance to change were identified as significant barriers to technology adoption in some cases. "Changing established ways of doing things can be challenging, especially in organizations with a strong resistance to change," noted one participant. "We encountered resistance from some employees who were hesitant to embrace new technologies and processes, which slowed down our adoption efforts." 4.5 Outcomes and Implications Despite these challenges, participants reported a range of positive outcomes and implications resulting from technology adoption in their ventures. Improved efficiency, increased productivity, and enhanced competitiveness were commonly cited as benefits of technology adoption. For example, one participant discussed how technology had enabled them to streamline their operations and scale their business more effectively. "Technology has allowed us to do more with less," they explained. "By automating manual processes and leveraging data analytics, we've been able to increase our productivity and stay ahead of the competition." Similarly, another participant highlighted the impact of technology on their ability to innovate and differentiate their offerings in the marketplace. "Technology has been a game-changer for us in terms of innovation," they stated. "By embracing emerging technologies and experimenting with new ideas, we've been able to develop unique products and services that set us apart from competitors." Moreover, participants emphasized the broader societal impact of technology adoption in entrepreneurship, including job creation, economic growth, and social change. "Technology has the potential to create new opportunities and empower individuals and communities," remarked one participant. "By harnessing the power of technology for good, entrepreneurs can drive positive change and make a meaningful difference in the world." 5. Discussion The discussion section synthesizes the findings of the study and provides insights into the implications for theory, practice, and future research on technology adoption in entrepreneurship. By examining the motivations, processes, outcomes, and challenges of technology adoption among entrepreneurs, this section offers a comprehensive analysis of the complex interplay between technology and entrepreneurship in today's digital economy. First and foremost, the findings of the study highlight the multifaceted nature of technology adoption in entrepreneurship, revealing a diverse array of economic, strategic, and personal motivations that drive entrepreneurs' decisions to integrate technology into their Page 9/13 ventures. Economic considerations, such as the desire to enhance efficiency, reduce costs, and increase profitability, emerged as primary drivers of technology adoption, reflecting entrepreneurs' pragmatic concerns and business imperatives. Similarly, strategic motivations, including the pursuit of growth, scalability, and market competitiveness, played a significant role in shaping entrepreneurs' technology adoption efforts, underscoring the strategic importance of technology as a tool for achieving long-term success and sustainability. Moreover, personal motivations, such as passion for innovation, autonomy, and social impact, were identified as powerful drivers of technology adoption, highlighting the role of individual values, beliefs, and aspirations in shaping entrepreneurial behavior and decision-making. The findings also shed light on the processes and outcomes of technology adoption in entrepreneurship, revealing a dynamic and iterative journey characterized by experimentation, learning, and adaptation. Participants described a series of stages, decisions, and actions involved in the technology adoption process, including awareness, evaluation, trial, and adoption, highlighting the complexity and uncertainty inherent in navigating technological change in entrepreneurial ventures. Despite the challenges and barriers encountered along the way, participants reported a range of positive outcomes and implications resulting from technology adoption, including improved efficiency, increased productivity, enhanced competitiveness, and broader societal impact. These findings underscore the transformative potential of technology to drive innovation, growth, and positive change in entrepreneurship, while also highlighting the need for careful planning, resource allocation, and organizational support to maximize the benefits of technology adoption and mitigate potential risks and challenges. The discussion also touches upon the broader implications of the study for theory, practice, and policy in the field of technology adoption in entrepreneurship. From a theoretical perspective, the findings contribute to our understanding of the motivations, processes, and outcomes of technology adoption among entrepreneurs, enriching existing frameworks and theories in the fields of entrepreneurship, innovation management, and information systems. By elucidating the complex interplay of economic, strategic, and personal factors that influence technology adoption decisions, the study provides a nuanced perspective on the dynamics of technological change in entrepreneurial ventures, offering valuable insights for researchers seeking to advance theory and knowledge in this area. From a practical standpoint, the findings offer actionable insights and recommendations for entrepreneurs, policymakers, and practitioners seeking to support and promote technology adoption in entrepreneurship. By highlighting the diverse array of motivations driving technology adoption, the study underscores the importance of aligning technological investments with entrepreneurial goals, values, and aspirations. Moreover, by identifying common challenges and barriers to technology adoption, such as technical complexity, resource constraints, and resistance to change, the study provides guidance on how to overcome these obstacles and foster a supportive ecosystem for technology-enabled entrepreneurship. From a policy perspective, the findings underscore the need for targeted interventions and initiatives aimed at promoting digital literacy, access to resources, and supportive infrastructure to enable entrepreneurs to harness the full potential of technology for innovation and growth. 6. Conclusion Page 10/13 In conclusion, this study has provided valuable insights into the motivations, processes, outcomes, and challenges of technology adoption in entrepreneurship. Through in-depth interviews and thematic analysis, the study has shed light on the complex interplay of economic, strategic, and personal factors that drive entrepreneurs' decisions to integrate technology into their ventures. The findings reveal that economic considerations, such as the desire to enhance efficiency, reduce costs, and increase profitability, play a significant role in driving technology adoption among entrepreneurs. Similarly, strategic motivations, including the pursuit of growth, scalability, and market competitiveness, influence entrepreneurs' technology adoption efforts. Moreover, personal motivations, such as passion for innovation, autonomy, and social impact, are powerful drivers of technology adoption, reflecting entrepreneurs' values, beliefs, and aspirations. The study also highlights the dynamic and iterative nature of the technology adoption process, characterized by experimentation, learning, and adaptation. Despite the challenges and barriers encountered along the way, entrepreneurs reported a range of positive outcomes resulting from technology adoption, including improved efficiency, increased productivity, enhanced competitiveness, and broader societal impact. The implications of the study extend to theory, practice, and policy in the field of technology adoption in entrepreneurship. From a theoretical standpoint, the findings contribute to our understanding of the dynamics of technological change in entrepreneurial ventures, enriching existing frameworks and theories in entrepreneurship, innovation management, and information systems. From a practical perspective, the study offers actionable insights and recommendations for entrepreneurs, policymakers, and practitioners seeking to support and promote technology adoption in entrepreneurship. 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E3S Web of Conferences 291, 05011 (2021) SDGG 2021 https://doi.org/10.1051/e3sconf/202129105011 Gender markers in the space of a Russian university and their role in assessing the quality students’ life (on the example of regional universities) Anastasia Babaeva*, Artem Kluev, Julia Marinina, Anna Merzlyakova, and Julia Shlykova Kozma Minin Nizhny Novgorod State Pedagogical University, Ulyanov str, 1, 603000 Nizhny Novgorod, Russia Abstract. The article is devoted to the analysis of subjective well-being which is concerned as a constituent element of the integral concept of the quality of life. Theoretical conclusions of the study are based on empirical data from sociological research. The study focuses on Russian students and their well-being in Russian educational environment. The authors examine the influence of gender aspects on the students’ comfort level. Gender markers which function in business and personal communication at the university are investigated in vertical and horizontal cross-sections. Some peculiarities in the assessments given by Russian students on the subject of gender-sensitive social practices are noted. The main factors that determine the assessment of gender markers presence at the university are revealed. The lack of formed gender competence among Russian students is also revealed. The influence of gender markers on student satisfaction with studying process at the university is determined. 1 Introduction The concept of the quality of life, which the UN has put as the basis for comparing and assessing the socio-economic development of countries, is integrative. It includes indicators that reflect physical, mental, emotional and social health of a person. The World Health Organization (WHO) proposes to interpret the term “quality of life” as an individuals’ perception of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards and concerns". Thus, this concept includes the following indicators: living standards, level of governmental and social support of individual interests and goals, compliance with expectations. Some countries consider as part of the concept the opportunity to demonstrate various kinds of activity and enjoy them [1]. The complexity of using the concept of quality of life (and its derivatives) is determined by the fact that there is no single, generally accepted interpretation of it. However, a set of fundamental indicators that appear in the UN reports allows us to make some conclusions: 1) consideration of the quality of life of a country is directly * Corresponding author: dff1890@yandex.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 291, 05011 (2021) SDGG 2021 https://doi.org/10.1051/e3sconf/202129105011 related to its prospects for socio-economic development; 2) the study of the quality of life raises the problem of national security. The phenomenon of national security is associated with the resistance of socio-economic, political and cultural systems to destabilizing external and internal impacts [2]. The structure of the concept in question includes, first of all, objective indicators: life expectancy, absence of threats to life and health, level of unemployment, volume of consumption of goods and services, access to cultural values and education, etc. These are the metrics that are quantified and described. The key indicators here are the volume and dynamics of gross domestic product and the human development index (HDI). The second block of indicators consists of subjective factors - the index of happiness, or satisfaction with one's life. These are qualitative indicators, they are more difficult to analyze, but in combination with them, the actual data reflect the real picture of the quality of life [3]. The purpose of this report is to analyze the subjective factor, more specifically satisfaction with the life of a particular social group - student youth. We focus on the degree of comfort of learning process for higher education students. The study also takes in consideration the functioning of gender markers in university environment. In this regard, the study of gender markers in the environment of Russian university raisis the range of problems associated with a) the demographic situation (the issue of Russian youth reproductive health and their reproductive behavior); b) social relations and the level of conflict in society (lack of gender asymmetry and harmonious relations in the educational environment); c) cognition of the world and personal development, carried out through the institution of education (equal access to different degrees of education in various specialties, regardless of gender). Such studies are becoming more and more important for several reasons. Firstly, the modern stage of social development is characterized by the transition from a production economy to a knowledge economy. This means that knowledge, values and experience are becoming the leading factor in socialization. So education can be interpreted as a key to the future, the way to produce a new human format. Everything is important here: the content of the courses (what is broadcast), the teaching method (how it is broadcast), the equipment of the classrooms and the organization of classes (where it is broadcast). Researchers shouldn’t lose sight of extracurricular communication principles and general principals of learning process organization. The role of educational institutions in modern Western society, which includes Russia, is also increasing because traditional institutions - the church and the family are loosing their importance. At the same time the concept of lifelong education is gaining popularity. As a result, we get a social institution that accompanies a person throughout his life. Individual’s existence largely depends on what he or she receives at University: what knowledge and values. It also depends on how comfortable an individual feels at University as well. Secondly, there is growing interest in the study of the gender aspects of educational practices, it is due to the increasing technocratization and informatization of social practices [4]. New spheres and forms of employment are emerging. For example, to apply for a job candidates have to meet with some new requirements for their strength or endurance. So the question arises, how does this socio-economic context affect basic, biological parameters of a person and the assessment of his productivity or relevance? Is Western society moving into the phase of posthuman, in particular androgynous existence? How do educational institutions meet these challenges? [5]. Another reason for the problem to remain burning is the desire of society, in the form of the State, to determine the main vectors, principles and direction of social policy on family and demography issues. Russia was the first state in history to develop a whole range of 2 E3S Web of Conferences 291, 05011 (2021) SDGG 2021 https://doi.org/10.1051/e3sconf/202129105011 measures and even the ideology of women's emancipation. Modern researchers note [6] that cultural practices (the political program started by the Bolsheviks) had the following consequences: the raise of women’s authority (as a mother and a worker) in Soviet society led to the decrease of male importance. Often these practices in the USSR were formalized, for example, there was a quota for the presence of women in the Supreme Soviet of the USSR, but seats were distributed mainly between economy, agriculture and factory production sectors. Soviet women, representatives of intellectuals and managers, were not included in the Supreme Soviet. However, the development of the social sphere in the USSR, the protection of motherhood and childhood led to significant success of Russian society in solving the women's issue. Nowadays modern Russian society in the new conditions of its existence is looking back on the traditional ways of solving not only the female, but also the male issue. The male issue becomes increasingly significant for Western society in the XXI century. The fourth reason to study gender issues in social practices in general, and in education in particular, is the unprecedented global employment situation caused by the pandemic. According to the World Happiness Report, Section 7, Work and Wellbeing During COVID-19: Impact, Inequality, Resilience, Future of work ... it is found out that, “Globally, four in ten employed women work in sectors that were hard-hit by COVID-19, including travel, retail, food, accommodation, and services”. Consequently in the nearest future education programs are expected to be streamlined in the context of surplus / lack of qualified personnel in certain sectors of the economy, which could potentially affect the ratio of representatives of both sexes in the number of students, and subsequently increase gender asymmetry in the labor market [7]. However, in the same report the following trend is described: the gender gap is getting smaller, fathers have also increased time spent on childcare and housework since the beginning of the pandemic. During the pandemic both sexes had to stay at home equally. Nevertheless it is still difficult to say whether the trend will develop in the future, but it deserves to be taken in consideration. Studies of gender impact in education have both theoretical significance and practical relevance [8]. The development of a balanced social gender policy, as well as the assessment of its effectiveness, require scientific approach. 2 Materials and methods Theoretically, this report is based on the results of empirical research carried out on the basis of Minin University. Quantitative methods (questionnaires) and qualitative methods (focus groups, in-depth interviews) are used. The first stage of studies was completed in 2017. These studies were focused on the analysis of the degree of citizens’ satisfaction with the development of Russian province cultural environment (on the example of Nizhny Novgorod). The focus on Nizhny Novgorod is determined not only by the location of the research base, but also by the status of the city in the Russian Federation. Nizhny Novgorod is a metropolis with a population of over 1,250,000. The city itself is located in the central part of Russia, it is an industrial and cultural center. These circumstances determine high standards of cultural institutions demanded by residents and high level of critical assessments of quality of life in general. The second stage of research is devoted to fixing and determining the functioning of gender markers in the educational environment of the university. These studies have been conducted since 2018. The object of the second stage is full-time students of universities, mainly in Nizhny Novgorod, as well as the teaching staff of higher education organizations. 3 E3S Web of Conferences 291, 05011 (2021) SDGG 2021 https://doi.org/10.1051/e3sconf/202129105011 The focus on the university space was largely determined by the thesis: the university is a corporation, where an atmosphere of freedom of exchange of opinions is cultivated for the development of science and the launch of innovations, the formation of cohesion and tolerance. Since the studentship is a group that includes representatives of various territorial communities (regional and district centers, urban-type settlements, villages), the results of the research carried out as a whole can be extrapolated to the Russian reality with some limitations. We assume that the transfer of results to the regions, primarily the North Caucasus, as well as Moscow, St. Petersburg and the Kaliningrad region, will be irrelevant. 3 Results and discussions Since the focus of research interests is quite wide and changes over time, on the basis of Minin University, author's questionnaires and scenarios for qualitative research developed by a working group are used, which include both teachers and students, are used. In the questionnaires, the questions are not grouped into blocks, but in order to achieve greater objectivity, they are arranged separately. The texts of questionnaires and interviews contain questions that are aimed at identifying the general gender identification of the respondents, establishing opinions about the initial parameters that determine the identification of a person by gender; the degree of influence of various factors and actors in the cultivation of types of masculinity and femininity in society. The central place in the questionnaires is given to questions aimed at fixing and analyzing the functioning of gender markers at the university. The developed methods make it possible to record the opinion of the respondents about: a) the peculiarities of male and female behavior, approved / disapproved behavior in the society; b) the presence of gender equality / inequality in social practices in general, including in the educational environment of the university; c) the presence of gender segregation practices at the university; d) the sensitivity of the university environment to various manifestations of gender and gender characteristics; e) willingness to oppose practices / maintain practices of non-equilibrium gender relations, etc. And most importantly, the content of teaching materials allows us to explore the wellbeing of students in a gender-sensitive educational environment. The results obtained in the course of the study, at first glance, turn out to be contradictory. The results indicate the presence of gender markers, but the reaction to their presence in the university is ambiguous. Let us dwell in more detail on those moments that can directly testify that students feel at a gender-tinged university. Attention is drawn to the fact that the respondents almost unanimously note that the influence of gender markers in the university is less noticeable than in other spheres of life. Many respondents, noting the (relative) gender / gender neutrality of the university, compared the university with a family, school, sports organizations. Based on this comparison, individual respondents (about a tenth of the respondents) concluded that the sex and gender characteristics of a person do not matter at all at a university. Explaining their position, students and teachers most often explained this by the fact that at the university much depends on knowledge, ability to work and the desire to study / engage in scientific activity / be realized in a different way. However, up to about 18% within the framework of quantitative methods and more than half of the interviewed students noted that they themselves faced or witnessed how gender played a special role, for example, in the formulation of an assessment: the teacher 4 E3S Web of Conferences 291, 05011 (2021) SDGG 2021 https://doi.org/10.1051/e3sconf/202129105011 overestimated or underestimated the requirements for abilities (physical, mental, etc. .). Let us illustrate the thesis with an excerpt from an in-depth interview: “Sometimes the teachers ask them less [young men]. If a girl submits a written work, then they demand that everything be neat: in a file, without crumpled leaves ... and if the boys hand over, then, thank God, even though they have passed, they are already great, they don't even look at the design ... "(3rd year student Faculty of Humanities, Nizhny Novgorod). With regard to teachers, it is worth noting that, for their part, they also recognize the fact of a change in the volume of requirements based on gender, but only in specialized disciplines. For example, such a practice is recognized in the field of such training profiles as Physical Culture and Sports, Philosophy (interviewed by the Nizhny Novgorod NRD). It should be noted that the teachers, recognizing the facts of an unbalanced relationship, sought to comment on the position, for example: “How could it be otherwise? I can't ask boys and girls alike! They have different standards ... it's biology. " It is noteworthy that the survey of students of Nizhny Novgorod universities in the 2018-2019 it is recorded the following point: 33.8% of respondents stated that they had witnessed situations where teachers discriminated against students on the basis of gender, and the most numerous cases were the facts of harassment by women teachers against girls 17.7%; 8.7% of respondents pointed to harassment of girls by male teachers, the remaining percentage accounted for the facts of harassment of young men by teachers, and women teachers (4.4%) again bypassed men. Thus, the female part of the studentship is recognized as the most discriminated group at the university, and the female part of the teaching staff is the most discriminatory. At the same time, analysis of the same questionnaire shows that 73.3% of respondents find the gender of the teacher to be unprincipled, 13.6% note that it is more comfortable for them to study with a teacher of their own gender, and 12.1% are the opposite. It is noteworthy that any methodology aimed at identifying gender markers in the university space (the first questions of the questionnaire, interviews, throwing into the work of focus groups) initially records the answers, the content of which is the denial of gendersensitive practices. There are minimal respondents who initially state the presence of gender in the university. However, as they progress through the questionnaires, respondents designate the practices of non-equilibrium gender-based attitudes that take place within the walls of the university: jokes related to the behavior or characteristics of a particular gender, separation (independent or at the initiative of a teacher) based on gender when solving professional or educational tasks. Analyzing the results of the study, we have to admit that even the facts of the presence of gender markers in the space of a Russian university are ascertained by the respondents with difficulty. On the one hand, a sophisticated eye could recognize in this the intent and consistent application of veiling practices. Sex / gender (here we do not distinguish between biological and social sex), indeed, refers to the fundamental codes in culture. Having undergone coding at the early stages of the formation of culture, the dichotomy of sex in Russian social practices still persists. In Russia, within the framework of the official line, 2 sexes are recognized, in this respect the concept of “gender” should rather be interpreted in the meaning of the social functions of sex, that is, as a cultural superstructure over biology. Our research shows that the perversions that exist in the "backyards" of culture while only strengthening the official line. On the other hand, we have to admit that Russian students often simply lack so-called gender competence, which is understood as the ability to avoid being “the subject and object of situations of gender inequality”. In fact, being a gender-competent person means having an understanding of discriminatory practices based on gender, having the ability not to initiate such practices, and resisting sexist influences. Our research has demonstrated the 5 E3S Web of Conferences 291, 05011 (2021) SDGG 2021 https://doi.org/10.1051/e3sconf/202129105011 inability of students not only to counteract, but also to elementarily see such facts and evaluate them properly. In this case, we could put forward the thesis that the underdevelopment of gender competence is due to the absence of relevant problems in Russian society as a whole, especially if we recall the successes of the USSR in neutralizing gender discrimination. However, when to the question: “In your opinion, are there professions / areas of activity only for men and only for women”, the respondents answer in the affirmative and cite as an example pedagogy as a sphere of care that should traditionally be assigned to a woman, and what is more interesting - they recognize this as a normative practice, which they regard positively. Symptomatic in this case is not so much the fact that the respondents assigned pedagogy to women, but rather the making of a special value judgment on this matter. If the logical series is developed, a clear hierarchical ladder with rigid segregation indicators and corresponding practices is easily built. Translating the results of our research into the category of quality of life requires us to correlate quantitative indicators with subjective assessments of the subject, and how comfortable it is for students to exist in a situation of having, for example, different levels of requirements or principles of organizing classes based on gender. Studies show that the studentship is very loyal to such things. Thus, quantitative and qualitative methods demonstrate the presence of gender markers in the university environment, while about 70 percent of research participants either determine their presence by basic, normative things about which there is no need to reflect; or they characterize it positively, explaining the facts of a non-equilibrium relationship with the concern of the state, an established tradition, a biological given. Another 20 percent “find it difficult” to assess. Thus, those who are uncomfortable under the current conditions are in a clear minority. At the same time, it should be noted that students who feel unhappy in the university space solely on the basis of gender have not been identified. However the total number of respondents is those who see discriminatory practices and feel their negative impact, the number is insignificant in comparison with other countries. 4 Conclusion Thus, based on empirical data, it can be stated that the majority of Russian students feel comfortable in the gender-marked educational environment of the university. It is too early to speak about the impact of the pandemic on the gender well-being of the academic sphere - more research is needed here, and it will take time for educational institutions to react to the process. As for the socio-cultural and political contexts of the development of the topic of gender in the educational and academic environment of our country, it is absolutely necessary to take into account the influence of the traditions of each region, which ultimately determines the specifics of the Russian situation. Perhaps Western researchers would assess such a situation as a cognitive gap and an epistemological challenge, but the student community, realizing the presence of an imbalanced relationship based on gender in education, legitimizes the established practices. And since the university opens the door to an already established personality with a formed value paradigm, it would not be an exaggeration to say that not only students, but Russian reality as a whole is quite loyal to the practice of non-equilibrium gender relations. References 1. Encyclopedia Britanica, https://www.britannica.com 6 E3S Web of Conferences 291, 05011 (2021) SDGG 2021 https://doi.org/10.1051/e3sconf/202129105011 A. N. Zubets, Russian and international approaches to measuring the quality of life (2020) 3. R. Costanza, B. Fisher, S. Ali, C. Beer, L. Bond, R. Boumans, D. E. Gayer, Ecological Economics, 61(2-3), 267 (2007) 4. M. Castells, The Rise of the Network Society, The Information Age: Economy, Society and Culture. Oxford: Wiley-Blackwell, I (2010) 5. S. Kabisch, F. Koch; E. Urban Transformations: Sustainable Urban Development Through Resource Efficiency, Quality of Life and Resilience. Book series: Future City, 10, 1 (2018) 6. O. A. Khasbulatova, Russian Gender Policy of the 20th Century: Myths and Realities (2005) 7. M. Cotofan, J.-E. De Neve, M. Golin, M. Kaats, G. Ward, Work and Well-being during COVID-19: Impact, Inequalities, Resilience, and the Future of Work, https://worldhappiness.report 8. H. Wu; X. Ran; T. Zhang, et.al., Value in Health, 21, S227 (2018) 9. V. V. Teslinov, et.al., Substantiation of the Russian concept of lifelong education for adults (2014) 10. A. I. Subetto, Theory of quality of life (2017) 11. A. V. Babaeva, E. V. Kuznetsova, N. V. Shmeleva, M. Yu. Shlyakhov, Prospects for Science and Education, 5(41), 117 (2019) 2. 7 

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Case Report Thyrotoxic Periodic Paralysis: A Spine Consultation Abstract Samuel J. Mease, MD Michael J. Faloon, MD Conor J. Dunn, MD Stuart Changoor, MD Nikhil Sahai, MD Awais K Hussain, MD Arash Emami, MD From the Department of Orthopaedic Surgery, St. Joseph’s University Medical Center, Paterson, NJ (Dr. Mease, Dr. Faloon, Dr. Dunn, Dr. Changoor, Dr. Sahai, and Dr. Emami), and the Department of Orthopaedic Surgery, University of Illinois College of Medicine, Chicago, IL (Dr. Hussain). Correspondence to Dr. Emami: Emamiresearch@gmail.com None of the following authors or any immediate family member has received anything of value from or has stock or stock options held in a commercial company or institution related directly or indirectly to the subject of this article: Dr. Mease, Dr. Faloon, Dr. Dunn, Dr. Changoor, Dr. Sahai, Dr. Hussain, and Dr. Emami. JAAOS Glob Res Rev 2019;3: e10.5435/JAAOSGlobal.D.18.00082 DOI: 10.5435/JAAOSGlobal-D-18-00082 Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Orthopaedic Surgeons. This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. As a consultant, the orthopaedic spine surgeon is often asked to evaluate patients with acute-onset extremity weakness. In some cases, patient’s deficits can be attributed to nonspinal pathology; therefore, it is important to be aware of nonorthopaedic diagnoses when evaluating these patients. We report a case of thyrotoxic periodic paralysis that was initially confused by the consulting service with spinal pathology. A 32-year-old Hispanic man presented to our emergency department with rapid onset of lower extremity weakness. The consulting team ordered CT of the cervical and lumbar spine, as well as MRI of the lumbar spine which was aborted due to the patient’s worsening tachycardia and chest pain. The spine service was subsequently consulted to evaluate the patient. Review of the metabolic panel revealed a low potassium, and additional testing led to the eventual diagnosis of thyrotoxic periodic paralysis. After correction of the patient’s potassium, his weakness rapidly resolved, and no additional spinal workup was pursued. We describe this patient’s presentation and outline the differential diagnosis for acute, nontraumatic extremity weakness, including both orthopaedic and other medical causes, that the spine surgeon should be aware of when evaluating patients with extremity weakness. T he acute presentation of extremity weakness is a potentially emergent situation, which may result in consultation of the spine service. Although there are many pathologies of spinal etiology (including compression due to hemorrhage, infection, neoplasm, or disk herniation) that may result in the sudden onset of weakness without inciting trauma, the examining orthopaedic surgeon or neurosurgeon should consider other nonspinal etiologies in their differential diagnosis. Thyrotoxic periodic paralysis (TPP) is one such condition that, while rare, can result in notable sequelae if unrecognized and untreated. TPP is a condition characterized by thyrotoxicosis, hypokalemia, and paralysis, most often occurring in men of Asian or Hispanic descent.1,2 Although uncommon, it is increasingly reported in the United States because of the rising immigrant population. The paralysis associated with this condition most often affects the lower extremities. The hypokalemia results from an intracellular shift of potassium induced by thyroid hormone sensitization of Na1/K1 ATPase.1 Thyrotoxic Periodic Paralysis It is important for clinicians to be aware of the pathology and presentation of TPP because it often manifests similarly to another condition, familial periodic paralysis. Misdiagnosis can result in delay of appropriate treatment. Although both conditions may present with paralysis and hypokalemia, subtle differences, such as no family history of paralysis, hyperthyroid symptoms, male sex, and presentation between second and fourth decades of life, can aid in the diagnosis of TPP. Prompt diagnosis allows for definitive management of TPP, which includes nonselective beta-blockers and correction of hyperthyroidism.1,3 The following case report demonstrates a case of TPP with lower extremity paralysis for which the orthopaedic spine service was consulted. Case Report Clinical Course Our patient is a 32-year-old Hispanic man with no known medical history, who presented to the emergency department with a chief complaint of rapid onset of bilateral lower extremity weakness. He was initially evaluated by the emergency department physician and sent for a CT scan of his cervical and lumbar spine, as well as MRI of his lumbar spine. During the MRI study, he began to complain of severe substernal chest pain and became tachycardic and diaphoretic. Therefore, the study was terminated, with only sagittal T2 images having been obtained. At this point, orthopaedic spine consultation was placed, and the patient was evaluated by the orthopaedic service. The patient was anxious appearing. He reported rapid progression of bilateral lower extremity weakness, since its onset the evening before presentation, from which he was no longer able to 2 ambulate. He also reported heart palpitations, chest pain, and feelings of generalized anxiety. He denied any recent trauma, illness, or history of similar occurrence. He denied any paresthesias, fevers, chills, or constitutional symptoms. Examination, Laboratory Test Results, and Imaging On examination, he had profound motor deficits in his bilateral lower extremities, with 0/5 muscle strength in the L2-S1 distribution. In the upper extremities, he also had evidence of profound weakness, with approximately 3/5 motor strength in the C5T1 distributions. Sensation was intact globally. He was hyporeflexic in bilateral upper and lower extremities with no evidence of long tract signs including clonus or Hoffman’s sign. Perianal sensation was intact, although the patient’s rectal tone was decreased. Laboratory evaluation revealed potassium of 1.6 milliequivalents per liter (mEq/L) (normal: 3.5 to 5.0 mEq/L), and phosphorous of 1.7 milligrams per deciliter (mg/dL) (normal: 2.5 to 4.5 mg/dL). There were no other notable electrolyte abnormalities. The patient’s pulse was 139 beats per minute. ECG obtained after his aborted MRI demonstrated wideQRS-complex tachycardia (QRS interval 164 milliseconds). Amiodarone was administered after which the patient’s heart rate was well controlled and the QRS interval normalized. Subsequent thyroid function tests revealed hyperthyroidism and thyrotoxicosis (T4 = 18.4 micrograms per deciliter (mg/dL), T3 = 2.52 nanograms per milliliter (ng/mL), and thyroid stimulating hormone = 0.018 milliunits per liter (mU/mL)) (normal: T4 4.6 to 12 mg/dL; T3 0.8 to 2.0 ng/mL; TSH 0.4 to 4.0 mU/L, respectively). The CT and MRI imaging studies of the patient’s spine ordered before orthopaedic consultation demon- strated no evidence of compression or other notable abnormalities. Neurology and endocrinology consultation was recommended and obtained. Intravenous potassium was administered after which, on repeat evaluation by the orthopaedic team, the patient was found to have dramatic improvements of his previously appreciated motor deficits. Therefore, completion of MRI evaluation of the entire spine was deemed unnecessary, and care was deferred to medical subspecialties, with the orthopaedic team following closely. Management and Post– Emergency Department Course The patient was diagnosed with TPP, and endocrinology consultation was obtained. As per recommendations of the endocrinology team, the patient was placed on methimazole (10 mg every 8 hours) and propranolol (40 mg every 6 hours). Methimazole was then changed to 20 mg twice daily. The patient was also started on dexamethasone (2 mg intravenously every 8 hours) because of its role in inhibiting peripheral conversion of thyroid hormone from T4 to the more active T3 form.4 On final evaluation before discharge, the patient had fully recovered all deficits. Discussion This was a case of a 32-year-old Hispanic man who presented with acuteonset lower extremity weakness. The orthopaedic service was consulted for evaluation of the spine; however, imaging obtained before consultation demonstrated no evidence of acute spinal pathology. Laboratory studies demonstrated hypokalemia and elevated thyroid hormone, suggesting that the cause of the patient’s weakness was not spinal in nature. The constellation of symptoms, as well as laboratory and imaging studies, eventually led to the Journal of the American Academy of Orthopaedic Surgeons Samuel J. Mease, MD, et al diagnosis of TPP. The patient was treated with methimazole, propranolol, and dexamethasone, after which his presenting symptoms were completely resolved at the time of discharge. TPP is a condition that occurs in 0.1% to 0.2% of the hyperthyroid population in North America.5 It occurs more often in men than in women and is more common in Asian and Hispanic individuals.1,6 The characteristic triad of findings associated with TPP includes hypokalemia, hyperthyroidism, and proximal muscle paralysis. Additional laboratory abnormalities that may be present in patients with TPP are hypomagnesemia and hypophosphatemia, which, along with hypokalemia, may also contribute to muscle weakness.7-9 The paralytic attacks associated with TPP occur mostly at night and are precipitated by alcohol, high carbohydrate diet, and heavy exercise.10 These characteristics can be considered when differentiating TPP from other causes of acute-onset extremity weakness. The patient in this case was also found to have wide complex tachycardia at the time of presentation. ECG changes related to electrolyte abnormalities have been well described in the literature including ST segment depression with flattened T waves, sinus tachycardia, U waves, seconddegree atrioventricular blocks, sinus arrest, ventricular fibrillation, and ventricular tachycardia.11,12 Thus, TPP represents a potentially life-threatening condition necessitating prompt diagnosis and treatment. Unfortunately, the diagnosis of TPP is often delayed and can be confused with more familiar causes of paralysis.2 Such delays in diagnosis can be partially attributed to the subtlety of thyrotoxic symptoms and the unfamiliarity of the treating physician with this rare condition.2 Thus, it is not farfetched to suggest that the orthopaedic or neurosurgical spine service may be consulted by the November 2019, Vol 3, No 11 emergency department for a patient with TPP, as was the case in our patient’s initial management. Proper and prompt diagnosis through history, physical examination, and laboratory evaluation can prevent deterioration of the patient, unnecessary imaging, and allow the patient to receive appropriate treatment in a timely manner. When evaluating a patient with acute extremity weakness, the spine surgeon’s differential diagnoses should not only be limited to compressive spinal pathology but also include other etiologies, including inflammatory, neurodegenerative, and metabolic. Although spine surgeons may be confident in diagnosing some causes of weakness, mostly compressive in nature, they may not be comfortable with less commonly encountered conditions that may present without obvious compression on imaging. TPP is one such metabolic condition that presents with acute extremity weakness. Other conditions to be considered are myasthenia gravis, GuillainBarré syndrome, and botulism. Certain findings can help differentiate TPP from other conditions presenting with acute-onset weakness. In contrast to compressive causes of weakness, imaging studies in patients with TPP are normal. In TPP, proximal muscles are typically more severely affected than distal muscles. Mentation remains intact in patients with TPP, the respiratory muscles are spared, and the cranial nerves are not affected. Another important finding is that sensation is unaffected in TPP.2,5,6 Guillain-Barré syndrome is another cause of weakness that can be differentiated from TPP by its classic presentation of ascending paralysis and abnormal cerebrospinal fluid findings.13 Botulism is a toxin-mediated condition that presents as descending flaccid paralysis where the muscles of the head and neck are initially affected.14 In con- trast to TPP, both Guillain-Barré syndrome and botulism can affect the respiratory system and lead to respiratory distress. Although neuromuscular disorders such as myasthenia gravis and Lambert-Eaton syndrome can present with extremity weakness, these are the result of an autoimmune reaction affecting the neuromuscular junction and do not typically present with electrolyte abnormalities.15,16 TPP is often misdiagnosed as familial hypokalemic periodic paralysis (FHPP) in Western countries because of similarities in precipitating factors and the clinical pattern of paralysis.1 However, TPP usually presents later in life with thyrotoxic symptoms and abnormal thyroid function tests. In contrast to FHPP, patients with TPP typically lack a family history of paralysis. In unclear cases, a urine calcium to phosphate ratio of .1.7 is a sensitive and specific test for diagnosing TPP and differentiating it from FHPP.5 In conclusion, although not a disorder of spinal etiology, it is imperative that spine surgeons are aware of TPP as it is a life-threatening condition associated with acute-onset extremity weakness for which they may be consulted. Key findings in the history, examination, and laboratory evaluation can aid in prompt diagnosis, obviating the need for expensive imaging. Prompt diagnosis reduces time to appropriate treatment, which includes nonselective beta-blockers, potassium repletion, and addressing the thyrotoxicosis. As the immigrant population in the United States continues to rise, so does the incidence of this uncommon disorder compelling spine specialists to be informed of TPP and to include it in their differential diagnosis.17 References 1. Lam L, Nair RJ, Tingle L: Thyrotoxic periodic paralysis. Proc (Bayl Univ Med Cent) 2006;19:126-129. Thyrotoxic Periodic Paralysis 2. Manoukian MA, Foote JA, Crapo LM: Clinical and metabolic features of thyrotoxic periodic paralysis in 24 episodes. Arch Intern Med 1999;159:601. 7. Knochel JP: The pathophysiology and clinical characteristics of severe hypophosphatemia. Arch Intern Med 1977; 137:203. 3. Magsino CH, Ryan AJ: Thyrotoxic periodic paralysis. South Med J 2000;93: 996-1003. 8. Tinker TD, Vannatta JB: Thyrotoxic hypokalemic periodic paralysis: Report of four cases and review of the literature (2). J Okla State Med Assoc 1987;80:76-83. 4. Suwansaksri N, Preechasuk L, Kunavisarut T: Nonthionamide drugs for the treatment of hyperthyroidism: From present to future. Int J Endocrinol 2018;2018:5794054. 5. Tella SH, Kommalapati A: Thyrotoxic periodic paralysis: An underdiagnosed and under-recognized condition. Cureus 2015; 7:e342. 6. Chen YC, Fang JT, Chang CT, Chou HH: Thyrotoxic periodic paralysis in a patient abusing thyroxine for weight reduction. Ren Fail 2001;23:139-142. 4 12. Fisher J: Thyrotoxic periodic paralysis with ventricular fibrillation. Arch Intern Med 1982;142:1362. 13. Willison HJ, Jacobs BC, van Doorn PA: Guillain-Barré syndrome. Lancet 2016; 388:717-727. 14. Carrillo-Marquez MA: Botulism. Pediatr Rev 2016;37:183-192. 9. Woolery WA, Gharib H: Thyrotoxic hypokalemic periodic paralysis in a white man. South Med J 1991;84:1399-1401. 15. Gilhus NE: Myasthenia Gravis. N Engl J Med 2016;375:2570-2581. 10. Hsu YJ, Lin YF, Chau T, Liou JT, Kuo SW, Lin SH: Electrocardiographic manifestations in patients with thyrotoxic periodic paralysis. Am J Med Sci 2003;326: 128-132. 16. Schoser B, Eymard B, Datt J, Mantegazza R: Lambert–eaton myasthenic syndrome (LEMS): A rare autoimmune presynaptic disorder often associated with cancer. J Neurol 2017;264:1854-1863. 11. Ee B, Cheah JS: Electrocardiographic changes in thyrotoxic periodic paralysis. J Electrocardiol 1979;12:263-279. 17. Patel J, Wilches LV, Guerrero J: Thryrotoxic periodic paralysis: Diversity in America. J Emerg Med 2014;46:760-762. Journal of the American Academy of Orthopaedic Surgeons 

W4255264935.txt

Qeios · Definition, February 2, 2020 Ope n Pe e r Re v ie w on Qe ios Case Report Tabulation National Cancer Institute Source National Cancer Institute. Case Report T abulation. NCI T hesaurus. Code C142411. Lists of information that are organized in some fashion. Qeios ID: XW9VJF · https://doi.org/10.32388/XW9VJF 1/1 

W3134835414.txt

BRIEF RESEARCH REPORT published: 04 March 2021 doi: 10.3389/fvets.2021.651978 Serological Evidence of Exposure to Peste des Petits Ruminants in Small Ruminants in Rwanda Anselme Shyaka 1*, Marie Aurore Ugirabe 1 and Jonas Johansson Wensman 2* 1 School of Veterinary Medicine, College of Agriculture, Animal Sciences and Veterinary Medicine, University of Rwanda, Nyagatare, Rwanda, 2 Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden Edited by: Satoshi Sekiguchi, University of Miyazaki, Japan Reviewed by: Richard Anthony Kock, Royal Veterinary College (RVC), United Kingdom Arnab Sen, University of North Bengal, India *Correspondence: Anselme Shyaka a.shyaka2@ur.ac.rw Jonas Johansson Wensman jonas.wensman@slu.se Specialty section: This article was submitted to Veterinary Epidemiology and Economics, a section of the journal Frontiers in Veterinary Science Received: 11 January 2021 Accepted: 09 February 2021 Published: 04 March 2021 Citation: Shyaka A, Ugirabe MA and Wensman JJ (2021) Serological Evidence of Exposure to Peste des Petits Ruminants in Small Ruminants in Rwanda. Front. Vet. Sci. 8:651978. doi: 10.3389/fvets.2021.651978 The status of Peste des Petits Ruminants (PPR) in Rwanda is unknown, despite its prevalence in neighboring countries. A cross-sectional sampling of goats and sheep was carried out in five districts of Rwanda located closer to neighboring countries endemic to PPR. Serum samples were analyzed using a commercial ELISA, to detect antibodies to PPR virus (PPRV). Sixty-eight samples [14.8, 95% Confidence Interval (CI): 11.7–18.4] were seropositive for PPR, of which 17.4% (95% CI: 11.6–24.6; 25/144) were from sheep, whereas 13.6% (95% CI: 10.0–17.9; 43/316) were from goats. Seropositivity ranged from 8.9 to 17.3% (goats) and from 10.5 to 25.8% (sheep) in sampled districts. Seropositivity was slightly higher in males than females in both goats (15.7 vs. 12.4%) and sheep (17.7 vs. 17.1%), and were significantly marked in goats and sheep aged more than 15 months (goats: 17.9, 95% CI: 12.9–24.0; sheep: 22.2, 95% CI: 14.1–32.2) than those between 6 and 15 months (goats: 6.1, 95% CI: 2.5–12.1; sheep: 9.3, 95% CI: 3.1–20.3). Sampling was non-randomized and results are not representative of the true prevalence of PPR antibody in small ruminants. Thus, data does not allow to fully discuss the findings beyond the presence/absence certitude and the comparisons made must be interpreted with caution. The presence of specific antibodies to PPRV may, however, be linked to one or a combination of following scenarios: (1) prevalence and persistence of PPRV in sampled regions which would cause low level of clinical cases and/or mortalities that go unnoticed; (2) introduction of PPRV to herds through movements of livestock from neighboring infected countries, and/or (3) events of disease outbreaks that are underreported by farmers and veterinarians. In addition to strengthen veterinary surveillance mechanisms, further studies using robust sampling methods and integrating livestock and wildlife, should be carried out to fully elucidate PPR epidemiology in Rwanda. Keywords: small ruminants, Rwanda, PPR, seroprevalence, transboundary diseases INTRODUCTION Livestock diseases are recognized global threats to food supply and to livestock industry specifically (1). Peste des Petits Ruminants Virus (PPRV) is a member of the family Paramyxoviridae, genus Morbillivirus, species Small ruminant morbillivirus (2). It primarily affects goats and sheep, but also other domestic animals such as cattle, pigs and camels as well as various wildlife ungulates (3, 4), Frontiers in Veterinary Science | www.frontiersin.org 1 March 2021 | Volume 8 | Article 651978 Shyaka et al. PPR Seroprevalence in Rwanda through contact with infected animals, or indirectly through fecal and/or mucosal secretions (5). The disease caused, Peste des Petits Ruminants (PPR), is highly contagious and is characterized by acute clinical signs in goats and sheep, as well as in wild ruminants (6–8). PPR is associated with a case fatality rate of 15.5% (8) that can reach up to 80–100% in naïve herds (9). PPR is recognized as the most widely distributed infectious disease of domestic small ruminants and wildlife ungulates, and is endemic in most countries of Africa, Middle East and Asia (10). It can negatively impact countries’ economy and increase poverty in rural settings where small ruminants are mostly concentrated. In fact, PPR-associated losses are estimated at USD 1.2–1.7 billion annually and a third of this financial burden occurs in Africa (10). In addition, PPR constitutes a growing challenge to biodiversity and wildlife conservation (8, 11). PPR has affected most countries in East Africa since the last 5 decades and confirmation of the first outbreak in Sudan in 1971–1972 (12), followed by further outbreak reports from Ethiopia in 1989–1990 (13). In Uganda, the major PPR outbreak was reported in 2006–2008, in Karamoja region along with a similar report in neighboring Kenya (14, 15). However, previous reports had suggested presence of PPR through seroprevalence studies carried out in the 1980s in Uganda and Kenya (16), in the 2000s in Uganda (17) and an outbreak reported in Uganda in 2003 (18). In addition, antibodies to PPRV were also detected in Ugandan wildlife in 2004 (19), probably as a consequence of spillover of the virus from livestock. Tanzania had its first confirmation of PPR in 2008 (20), with retrospective serological evidence of earlier circulation (17) and PPR is currently considered endemic, including in Kagera and Kigoma regions close to Rwanda (21). In neighboring Burundi, a first outbreak of PPR occurred in December 2017 to February 2018 (22), but retrospective serological analysis detected antibodies to the virus in samples collected in early 2017. A more recent study highlighted circulation of PPRV in livestock and wildlife living in eastern DRC and western Uganda (19). This brief history shows that PPR has had endemic events in various regions of east Africa surrounding Rwanda, with periodic outbreaks and circulation of the virus in various susceptible animals including detection in wildlife. Phylogenetic analyses of circulating viruses, showed that PPRV lineage II, III, and IV are prevalent in DRC, Uganda, Tanzania, Kenya, and Burundi (22–26). Rwanda status vis-à-vis PPR is unknown (27). In fact, there has never been any empirical study to establish the prevalence of PPR in the country, despite its occurrence in the neighboring countries (9, 19, 22, 24, 28–31). The presence of this disease in the regional countries, transboundary movements of livestock passing through official and non-official entry/exit points and important wildlife species constitute potential factors for PPRV introduction in the country. In order to strengthen prevention mechanisms against PPR in Rwanda toward eradication of PPR by 2030, it is important to establish systems of surveillance as part of the stage 1 or “Assessment stage” of the Global Strategy for the Control and Eradication of PPR (10). An efficient surveillance stage would give insights on whether the disease is present and passes unnoticed, and provide information for the next steps toward the elimination of PPR. Such surveillance mechanisms Frontiers in Veterinary Science | www.frontiersin.org must adopt strategies for the control of PPR in susceptible domestic and wild animals in Rwanda, in order to establish presence, circulation and persistence of the virus. This study investigated the prevalence of specific antibodies to PPRV and aimed at providing baseline data that can be used by concerned regulatory bodies and stakeholders to scale up PPR investigation in Rwanda and set up adequate prevention measures. MATERIALS AND METHODS Ethical Approval Ethical approval for this study was obtained from the College of Agriculture, Animal Sciences and Veterinary Medicine, University of Rwanda (Ethical approval reference: 025/17/DRIPGS). Approved consent forms were distributed and signed prior to the interviews and sampling of animals. Sampling of animals was done following the protocols in conformity with the World Organization for Animal Health (OIE) Terrestrial Animal Health Code 2012 (use of animals in research and education). Area Description and Study Design Samples were collected from Bugesera, Kirehe, and Nyagatare districts of the eastern Province in Rwanda, and from Gicumbi and Musanze districts of the Northern Province (Figure 1). The five sites sampled are relatively close to borders with Uganda to the north, Tanzania to the east and Burundi to the south. This cross-sectional study was conducted during a period of 3 months, from January to March 2019. Non-probability convenience samples were collected in farms located in the study area, under guidance of local veterinarians. In addition, a small questionnaire was used to collect information related to animal sampled, herd management and general animal health at the farm and its surrounding. Goats and sheep, apparently healthy, non-vaccinated against PPR and having more than 6 months of age, were recruited into the study. To accurately estimate the age of goats and sheep, the age dentition method was used, according to methods described elsewhere (32, 33). To calculate the sample size, we used a recommended formula for estimating the adequate sample size in prevalence study (34). Thus, assuming a large and homogenous population size with an estimated 50% prevalence (P) that optimizes the sample size, with a confidence level set at 95% and ±5% precision, the formula 2 n = z P(1−P) recommended 385 samples. Finally, 460 blood d2 samples were drawn from jugular veins of apparently healthy goats and sheep, using Vacutainer needles and sterile plain tubes. Samples were allowed to clot overnight in order to maximize sera collection, which were harvested following a centrifugation at 3,000 rpm for 5 min. The sera were then stored at −20◦ C until screening was done. Screening of the Samples The screening for the presence of PPR was done to detect antibodies to the nucleoprotein of Peste the Petits Ruminants virus, using a commercial competitive ELISA (cELISA) kit (ID screen R PPR competition, IDvet Genetics, Grabels, France) according to the manufacturer’s instructions. The sensitivity and 2 March 2021 | Volume 8 | Article 651978 Shyaka et al. PPR Seroprevalence in Rwanda FIGURE 1 | Map of Rwanda districts highlighting sampling sites in East and North areas. The map shows administrative boundaries of districts and provinces. The red triangles highlight sampling sites in Nyagatare, Kirehe and Bugesera districts of the Eastern Province as well as Musanze and Gicumbi districts in the Northern Province. RESULTS specificity of this cELISA in sheep and goats, is estimated at 94.5 and 99.4%, respectively, compared to virus neutralization assay (35). The optical densities (ODs) were read using a Thermo ScientificTM MultiskanTM FC Microplate Photometer at a wavelength of 450 nm and the results were expressed as sample positivity percentage (S/N %). Samples were considered positive if the S/N % were ≤50%, negative if ≥60% or doubtful if it was between 50 and 60%. Since the sampled small ruminants had no clinical signs of PPR, doubtful results were finally considered as negative. Characteristics of Study Respondents The descriptions below for study participants and characteristics of farms and small ruminants sampled can be found in Table 1. In total, this study reached 57 households distributed in the 5 districts targeted by this research. Participants were composed of 19 females and 38 males (Table 1). The age of respondents ranged between 18 to 87 in females and 16 to 83 in males, with a mean of 44 and 48 years, respectively. Considering sampled households, 35 of the 57 interviewed (61.4%), reported that small ruminants were managed under a zero-grazing method, in which animals were stall-fed on grasses and food residues. In the remaining herds, 13 and 9 farmers reported to apply open-grazing and semi-zero grazing systems, respectively, in which the small ruminants were allowed to graze freely or go around grazing and get a supplement of food residues once back home. Of the 57 farms targeted, the biggest share (36 out of 57, representing 63.2%) was owning goats, whereas 9 (15.8%) farms had only sheep and 12 (21.1%) had both sheep and goats housed together at farm level. In interviewed farmers, majority (47 of the 57, 82.5%) reported that their animals Statistical Analyses The true prevalence in positive animals and herds was estimated by adjusting the apparent prevalence obtained from cELISA results to the sensitivity and specificity of the test, as described by Rogan and Gladen (36). In addition, in order to test for independence between two variables, univariable analysis was done using chi-square test. All the statistical analyses were carried out using R Statistical Software (version 3.6.3; R Foundation for Statistical Computing, Vienna, Austria). Frontiers in Veterinary Science | www.frontiersin.org 3 March 2021 | Volume 8 | Article 651978 Shyaka et al. PPR Seroprevalence in Rwanda months, whereas 21 (36.8%) reported events of small ruminant deaths in neighboring farms (Table 1). In total, 316 goats (201 females and 115 males) and 144 sheep (82 females and 62 males) were sampled. Based on age dentition, the goats were classified into three main categories of age: between 6 and 15 months (115 goats), 1.5–3 years (105 goats) and those being more than 3 years (96 goats). Similarly, age estimation in sheep showed that 54 were between 6 and 15 months, 53 were between 1.5 and 3 years, whereas 37 were over 3 years (Table 2). TABLE 1 | Characteristics of study respondents and farms sampled (n = 45). Variables District Gender Age quintiles (years) Education Experience in animal husbandry (years) Types of small ruminant owned Farming system Small ruminants disease history Small ruminants disease history in neighboring farms Frequency (%) Nyagatare 6 (10.5) Kirehe 18 (31.6) Bugesera 7 (12.3) Gicumbi 12 (21.1) Musanze 14 (24.6) Female 19 (33.3) Male 38 (67.7) <20 5 (8.8) 21–30 3 (5.3) 31–40 11 (19.3) 42–50 17 (29.8) >50 21 (36, 8) No formal education 17 (29.8) Primary 28 (49.1) Secondary 12 (21.1) <1 1 (1.8) 1–5 24 (42.1) 6–10 8 (14.0) >10 24 (42.1) Goats only 36 (63.2) Sheep only 9 (15.8) Goats and Sheep 12 (21.1) Zero-grazing 35 (61.4) Semi-zero-grazing 9 (15.8) Open grazing 13 (22.8) Occurrence of abortions at sampled farms 19 (33.3) Occurrence of death at sampled farms 24 (42.1) Report of abortions in neighboring farms 12 (21.1) Report of death in neighboring farms 21 (36.8) Seroprevalence of PPR A total of 14.8% (68/460) samples from small ruminants, including 17.4% (25/144) from sheep and 13.6% (43/316) from goats were seropositive for antibodies to PPRV (Table 3 and Supplementary Figure 1). After adjusting to the test specificity and sensitivity, the overall animal-level estimated true prevalence was 15.1% (95% CI: 12.0–18.9), whereas species-level estimated true prevalence was 13.9% (95% CI: 10.3–18.3) and 17.8% (95% CI: 12.2–25.3) in goats and sheep, respectively. Of the 57 farms sampled, 35 had at least one animal seropositive (61.4, 95% CI: 48.4–72.9), giving an estimated farm-level true prevalence of 64.8% (95% CI: 50.9–77.0). DISCUSSION Sheep and goats represented 26% of 58,580 metric tons of red meat that was produced in Rwanda in 2017 (37) and this figure is expected to raise to meet growing population. Small ruminants are mainly raised for income generation through sales, but also for meat, wool and manure used in crop fields. Information generated from this study show that diseases affecting small ruminant and causing deaths and/or abortions are prevalent in the sampled regions. However, due to inadequate veterinary services penetration in rural Rwandan regions, characterized by widespread of less qualified veterinary paraprofessionals (VPP), inadequate veterinary supervision of the VPP (38, 39) and unavailability of supporting laboratory services, diseases that caused abortions were not clearly identified and/or communicated to farmers. PPR is known to cause abortions at all stages of the pregnancy (40). Among possible differential diagnosis, Rift Valley Fever (RVF), another disease that causes abortions in affected animals (41), must be taken into consideration as a possible factor associated to the episodes reported by farmers. In fact, Rwanda has had its first outbreak of RVF declared in 2018 (42) and cases were mainly identified in the eastern region of the country, part of our study area. This study is the first one to report seroprevalence of PPR in Rwanda. Our laboratory analyses indicated an estimated overall true prevalence of 15.1% (95% CI: 12.0– 18.9) and seropositivity of 13.9% (95% CI: 10.3–18.3) and 17.8% (95% CI: 12.2–25.3) in goats and sheep, respectively. These findings of PPRV-specific antibodies circulating in goats and sheep sampled in various areas, constitute evidence of exposure to the disease. Further studies are needed to provide more insights on the epidemiology of PPR in Rwanda. For instance, other investigations should were obtained from local livestock markets and others (10/57, making up 17.5%) through various donations. Interestingly, all farmers reported not to observe any quarantine period prior to introduction of new animals in their herds. Sampled Animals and Occurrence of Small Ruminant Diseases On a period of 12 months, interviewed farmers reported occurrence of abortions in 19 of their farms, representing 33.3%, whereas 12 farmers (21%) indicated occurrence of abortion incidences in neighboring farms. According to the 19 farmers who experienced abortions, 18 cases occurred in goats whereas 1 case concerned sheep. Also, of the 12 abortion occurrences observed in neighboring farms, all were reportedly observed in goats. Moreover, 24 farms (42.1%) highlighted occurrence of death involving small ruminants at their own farms in the past 12 Frontiers in Veterinary Science | www.frontiersin.org 4 March 2021 | Volume 8 | Article 651978 Shyaka et al. PPR Seroprevalence in Rwanda TABLE 2 | Characteristics of small ruminants sampled. Characteristics Sex Age Goats Sheep Total Male 115 62 177 Female 201 82 283 Total 316 144 460 6–15 months 115 54 169 1.5–3 years 105 53 158 >3 years 96 37 133 Total 316 144 460 TABLE 3 | Seroprevalence of PPR in Small Ruminants according to various disease risk factors. Risk factors District Sex Age Farming system Goats Sheep Total No. of samples No. of positive samples Sero-prevalence % 95% CI Total No. of samples No. of positive samples Sero-prevalence % 95% CI Nyagatare 75 13 17.3 (9.6–27.8) 21 4 19.0 (5.4–41.9) Bugesera 59 8 13.6 (6.0–25.0) 44 6 13.6 (5.2–27.4) Kirehe 70 9 12.9 (6.1–23.0) 29 5 17.2 (5.9–35.8) Musanze 56 8 14.3 (6.4–26.2) 31 8 25.8 (11.9–44.6) Gicumbi 56 5 8.9 (3.0–19.6) 19 2 10.5 (1.3–33.1) Total 316 43 13.6 (10.0–17.9) 144 25 17.4 (11.6–24.6) Male 115 18 15.7 (9.5–23.6) 62 11 17.7 (9.2–29.5) Female 201 25 12.4 (8.2–17.8) 82 14 17.1 (9.7–27.0) Total 316 43 13.6 (10.0–17.9) 144 25 17.4 (11.6–24.6) 6–15 months 115 7 6.1 (2.5–12.2) 54 5 9.3 (3.1–20.3) 1.5–3 years 105 17 16.2 (9.7–24.7) 53 12 22.6 (12.3–36.2) >3 years 96 19 19.8 (12.4–29.2) 37 8 21.6 (9.8–38.2) Total 316 43 13.6 (10.0–17.9) 144 25 17.4 (11.6–24.6) Zero grazing 155 18 11.6 (7.0–17.7) 67 14 20.9 (11.9–32.6) Semi-zero grazing 37 6 16.2 (6.2–32.0) 20 2 10.0 (1.2–31.7) Open grazing 124 19 15.3 (9.5–22.9) 57 9 15.8 (7.5–27.9) Total 316 43 13.6 (10.0–17.9) 144 25 17.4 (11.6–24.6) of Congo (9, 19, 22, 24, 28, 29, 43). In addition to reports of major outbreak in the region, various retrospective serological analyses, showed positive antibodies to PPRV and confirmed the prevalence of the virus and its circulation before occurrence of all recent outbreaks in above countries (17, 19, 22). Therefore, Rwanda Veterinary Services should strengthen active surveillance mechanisms in order to fully investigate prevalence of the disease and adopt prevention measures before occurrence of large outbreaks in the country. Future studies on PPR in Rwanda should depict a clearer picture of the epidemiology of the disease in Rwanda. For example, due to limitations inherent to this study, some unanswered questions were for example, the difference in the distribution of PPR across various regions and possible contribution of animal age, sex, and husbandry to the occurrence of PPR. provide more information on nation-wide prevalence in susceptible domestic and wildlife animals, risk factors associated to PPR prevalence and phylogenetic characterization of circulating viruses in an attempt to determine origin, spread and distribution of various virus lineages and risk factors in Rwanda. Based on laboratory data from this cross-sectional study, we certainly can confirm the exposure of goats and sheep to PPRV in sampled regions. The comparisons of prevalence with regional findings, should be undertaken carefully. In addition, this study has estimated prevalence of PPR in goats and sheep using non-probability sampling methods. Therefore, it does not allow generalizing the findings at country and small ruminant population level. PPR is a well-known disease in the region as shown by endemic as well as epidemic events having been reported in Uganda, Tanzania, Burundi and Democratic Republic Frontiers in Veterinary Science | www.frontiersin.org 5 March 2021 | Volume 8 | Article 651978 Shyaka et al. PPR Seroprevalence in Rwanda Our data suggests a correlation between the age of the sampled animals and seroprevalence status within age groups. In fact, small ruminants of more than 15 months were more affected than younger ones. This finding is in conformity with other studies on PPR (28, 44) and can be explained by the facts that older animals have had more exposure time to the virus, especially if this is endemic in the region. In addition, older animals tend to move far from their home in search of greener pastures and water bodies. Some study participants (10 of the 57 interviewed, Table 1) reported that their small ruminants were acquired through livestock donating initiatives. Therefore, we cannot rule out the possibility that the livestock were seropositive to PPRV, when gifted to farmers. The presence of antibodies in younger animals is however suggestive of recent virus circulation in Rwanda and this should be investigated further. Phylogenetic investigations showed that the virus lineages II, III and IV are circulating in the region. Based on limited available sequences, the lineage III seems predominating in western Uganda, eastern DRC and Burundi with sequence similarities in some countries such as DRC and Burundi (19). In addition, the 2017 outbreak of PPR that occurred in various regions in Burundi, followed introduction of Boer goats from potentially infected regions of Uganda and the goats were transported through Tanzania suggesting transboundary movements as possible route of PPRV transmission (22). The regional virus circulation, added to report of PPRV at wildlifelivestock interface in Kabale, Kisoro and eastern DRC; regions close to Rwanda (19), puts an emphasize on the role of movements of livestock across transnational boundaries as well as the role of wildlife animals in the circulation and maintenance of PPRV in the region. Our findings suggest that PPR is prevalent in Rwandan regions close to neighboring countries and areas with recent PPR outbreak events. Rwanda is located in an area characterized by large livestock as well as wildlife populations. In addition, the region is known for important livestock trade between regional countries including Rwanda, and eastern part of DRC (45). The presence of livestock and wildlife and trade movements across countries, could have contributed to the introduction of PPR in north and west parts of Rwanda. Last but not least, Rwanda has experienced large movements of returning citizens from neighboring countries in 1994 and from Tanzania in 2007 as well as Burundian refugees in 2015 (46– 48). These movements of people and their livestock could have contributed to the introduction of PPR in various regions of Rwanda. Molecular epidemiology studies and analysis of transboundary livestock movements could shed more insights on PPR epidemiology in Rwanda and the region. As a preliminary report, this study has several limitations. First, although PPR has never been declared in Rwanda, samples were taken from places relatively close to the borders of the country with countries with known reports of PPR in past years. Due to possible more intense transboundary livestock movements in sampled areas than in other parts of the country, the prevalence found in this study may not necessarily reflect a country-large situation. To minimize this bias, the calculation of needed sample size, assumed a prevalence of 50% which is a condition that maximizes the sample size. Secondly, sampling Frontiers in Veterinary Science | www.frontiersin.org methods were non-randomized and only a small number of farms was reached. Therefore, without assumption of a homogenous population, the data presented may not be representative of the entire population of small ruminant farmers. Therefore, the current findings do not allow to calculate the true prevalence, analyse the risk factors or to compare prevalence across the study areas. Third, cross-sectional surveys are not suitable for detection of rare, non-endemic diseases such as PPR with an unknown status in Rwanda. Further studies are needed to collect representative evidence, informative for the eradication and control programs. In this regard, comprehensive studies using probabilistic sampling methods are recommended to investigate PPR at wildlifelivestock interface in Rwandan regions neighboring DRC, Uganda, Tanzania and Burundi. Such studies would help to follow up the occurrence of disease events in small ruminants, and would retrospectively collect evidence of possible endemicity of PPR. This is justified by the possibility of regional circulation of the virus along with livestock transboundary movements as hypothesized by previous studies (19, 22). Rwanda is home to natural parks and forests which may serve as PPRV hotspots at the interface of livestock and wildlife. Therefore, future studies in Rwanda and the region, must take into consideration the livestock and wildlife components, in order to fully understand the epidemiology of PPR. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. ETHICS STATEMENT The studies involving human participants were reviewed and approved by Ethical approval for this study was obtained from the College of Agriculture, Animal Sciences and Veterinary Medicine, University of Rwanda (Ethical approval reference: 025/17/DRIPGS). The patients/participants provided their written informed consent to participate in this study. AUTHOR CONTRIBUTIONS AS contributed to conceptualization, laboratory analysis, data curation, investigation, methodology, and writing original draft. MU participated in conceptualization, investigation, methodology, and manuscript editing. JW contributed to conceptualization, funding acquisition, supervision, review of the manuscript, and editing conception and design of the study. All authors contributed to the article and approved the submitted version. FUNDING This study was funded by the Swedish Research Council (Grant No. 2018-03956). 6 March 2021 | Volume 8 | Article 651978 Shyaka et al. PPR Seroprevalence in Rwanda ACKNOWLEDGMENTS SUPPLEMENTARY MATERIAL We would like to thank Mr. Elysé Ndizeye and Mr. Justin Rucamihigo for their participation in data collection during this study. We would like to thank the Ministry of Agriculture and Animal Resources through the Rwanda Agriculture and Animal Resources Development Board (RAB) for their support in accessing the National Veterinary Laboratory facilities. We are grateful to Dr. Karin Alvåsen from Swedish University of Agricultural Sciences, for valuable discussions. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets. 2021.651978/full#supplementary-material Supplementary Figure 1 | Percentage Inhibition (PI) distribution based on competitive ELISA results (ID screen® PPR competition, IDvet Genetics, Grabels, France). The distributions are shown by sampled locations (District). Positive samples have below 50% PI and are shown in the graphs with a dark dashed black line. REFERENCES 17. Karimuribo ED, Loomu PM, Mellau LSB, Swai ES. Retrospective study on sero-epidemiology of peste des petits ruminants before its official confirmation in northern Tanzania in 2008. Res Opin Anim Vet Sci. (2011) 1:184–7. 18. AU-IBAR. Pan African Animal Health Yearbook 2003. Nairobi, Kenya: Interafrican Bureau for Animal Resources and African Union (2003). 19. Aguilar XF, Mahapatra M, Begovoeva M, Kalema-Zikusoka G, Driciru M, Ayebazibwe C, et al. 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Heliyon 6 (2020) e03258 Contents lists available at ScienceDirect Heliyon journal homepage: www.cell.com/heliyon Review article A proposed reverse transcription mechanism for (CAG)n and similar expandable repeats that cause neurological and other diseases Andrew Franklin a, Edward J. Steele b, c, *, Robyn A. Lindley d, e a Medical Department, Novartis Pharmaceuticals UK Limited, 200 Frimley Business Park, Frimley, Surrey, GU16 7SR, United Kingdom Melville Analytics Pty Ltd, Melbourne, Vic, 3004, Australia CYO’Connor ERADE Village Foundation, Perth, WA, Australia d GMDxgenomics, Melbourne, Vic, Australia e Department of Clinical Pathology, Faculty of Medicine, Dentistry & Health Sciences, University of Melbourne, Vic, Australia b c A R T I C L E I N F O A B S T R A C T Keywords: Neuroscience CAG expansions Huntington's disease Error-prone DNA repair AID/APOBEC/ADAR deaminases DNA polymerase-eta Immunoglobulin somatic hypermutation The mechanism of (CAG)n repeat generation, and related expandable repeat diseases in non-dividing cells, is currently understood in terms of a DNA template-based DNA repair synthesis process involving hairpin stabilized slippage, local error-prone repair via MutSβ (MSH2–MSH3) hairpin protective stabilization, then nascent strand extension by DNA polymerases-β and -δ. We advance a very similar slipped hairpin-stabilized model involving MSH2–MSH3 with two key differences: the copying template may also be the nascent pre-mRNA with the repair pathway being mediated by the Y-family error-prone enzymes DNA polymerase-η and DNA polymerase-κ acting as reverse transcriptases. We argue that both DNA-based and RNA-based mechanisms could well be activated in affected non-dividing brain cells in vivo. Here, we compare the advantages of the RNA/RT-based model proposed by us as an adjunct to previously proposed models. In brief, our model depends upon dysregulated innate and adaptive immunity cascades involving AID/APOBEC and ADAR deaminases that are known to be involved in normal locus-specific immunoglobulin somatic hypermutation, cancer progression and somatic mutations at many off-target non-immunoglobulin sites across the genome: we explain how these processes could also play an active role in repeat expansion diseases at RNA polymerase II-transcribed genes. 1. Purpose of this article It is necessary to directly state our overarching philosophy and rationale up front. We are molecular and cellular immunologists interested in the reverse transcriptase (RT) mechanism of antigen-driven somatic hypermutation (SHM) of rearranged immunoglobulin (Ig) variable region genes (Franklin et al., 2004; Steele et al., 2004; Steele et al., 2006; Steele, 2009; Steele, 2016; Steele and Lindley, 2017, Steele, 2017; these papers should be consulted for the molecular details of the RT Ig SHM mechanism). Why should we be applying mechanisms implicated in Ig SHM to the molecular events that precipitate (CAG)n and related trinucleotide repeat (TNR) disease? Somatic hypermutation underpins the generation of diversity in adaptive immunity in response to antigenic challenge, and while this response has physiologic benefit, it can also have pathologic consequence (such as in the case of cancer progression). In our view the generation of (CAG)n and related TNR may be interpreted as representing as a dysfunctional ‘Ig-SHM-like’ response allowing the postulate that the same or similar molecular processes might be involved. Viewing the molecular generation of trinucleotide repeat diseases through this prism provides fresh perspective and we hope contributes to advancement of the field. Our approach thus differs from other investigators working within the traditional discipline of (CAG)n and related TNR expansion diseases. In coding regions, TNR expansions occur in-frame, thus naturally lending themselves to an alternative RT-based explanation for their genesis. This is the purpose of this “Hypothesis” article and review. We are not supplanting the existing molecular mechanisms but rather adding to them by providing plausible and testable explanations for TNR expansions in RNA Pol II-transcribed regions arising from plausible RT processes transposed from our understanding of the molecular immunology of Ig SHM phenomena which may involve DNA synthesis opposite the pre-mRNA template of the target gene (Figures 1 and 2). We can say this now because we have shown that C-site and A-site off-target ‘Ig SHM-like’ mutagenic responses mediated by DNA and RNA deaminases (adenosine deaminases * Corresponding author. E-mail addresses: e.j.steele@bigpond.com, ejsteele@cyo.edu.au (E.J. Steele). https://doi.org/10.1016/j.heliyon.2020.e03258 Received 28 May 2019; Received in revised form 26 September 2019; Accepted 15 January 2020 2405-8440/© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). A. Franklin et al. Heliyon 6 (2020) e03258 Figure 1. Reverse transcriptase mechanism of immunoglobulin somatic hypermutation. Adapted from previous papers and as discussed in Box 1 and in the text (Franklin et al., 2004; Steele et al., 2006; Steele 2009, 2016; Steele and Lindley 2010, 2017; Lindley and Steele, 2013). Primary references can be found in these papers. The mechanism is an adaptation of the target site reverse transcriptase (TSRT) process first described in Luan et al. (1993). Independent confirmation (Box 2) that human DNA polymerase-η is a reverse transcriptase (RT) has been published recently by Su et al. (2017, 2019). The steps are: A. Modifications of DNA and pre-mRNA sequences at transcription bubbles via C-to-U and A-to-I deamination events, RNA polymerase II misincorporations (Kuraoka et al., 2003). into the pre-mRNA; B. DNA polymerase-η RT-priming complex; C. cDNA synthesis; D. cDNA strand invasion and heteroduplex formation; E. Resolution of the heteroduplex prior to DNA replication and cell division (MacPhee, 1995). Shown in A is the role of the RNA exosome at transcription bubbles in allowing access of AID to unpaired C-sites in the RNA:DNA hybrid (Basu et al., 2011). The key C-to-U deamination step at CAG repeats, from A. to B., is expanded in Figure 2. See Box 1 for further explanation. Black lines, DNA. Red lines RNA. Blue lines, cDNA. NTS, non-transcribed strand. TS, transcribed strand. McMurray, 2017). Thus we are advancing our model as a potential additional general mechanism for expandable TNR repeats in the main TNR expansion diseases in non-dividing cells in the brain – especially, those TNR expansion diseases involving TNRs that can form imperfect yet stabilized snap-back Watson-Crick base-paired hairpin structures on the pre-mRNA template (see Figure 3). So, in addition to (CAG)n, this would also apply particularly to (CTG)n and (CGG)n expansion diseases. It may also apply to the long GAA tract expansions of Friedreich's ataxia (FRDA), because more conventional DNA slippage could occur during replication involving the far weaker Hoogsteen (non-Watson-Crick) base pairing that would help create stabilized hairpins/R-loops (as listed in Table 1 of Usdin et al., 2015). So the model advanced in Figures 3 and 4 is most directly applicable, in terms of Watson-Crick base pairing, to (CAG)n codon expansions, but it could also apply to expansions affecting untranslated regions (UTR), such as 30 -UTR (CTG)n and 50 -UTR (CGG)n acting on RNA [ADARs], activation-induced deaminase [AID] and apolipoprotein B mRNA editing catalytic polypeptide-like proteins or APOBECs) appear to be operative also across the genome during cancer progression (Steele and Lindley 2010, 2017; Lindley, 2013; Lindley and Steele, 2013; Lindley et al., 2016; Mamrot et al., 2019). In our view, an understanding of these non-Ig off-target ‘Ig SHM-like’ mutagenic responses is related to our proposal here. That is, a molecular mechanism of TNR expansions may well involve pre-mRNA intermediates, as has been implicated to explain the molecular mechanism of Ig SHM in adaptive immunity and Ig SHM-like mechanisms in cancer progression. 2. TNR expansion diseases The current understanding of the molecular mechanism responsible for the expansion of DNA repeat sequences is incomplete (Polyzos and 2 A. Franklin et al. Heliyon 6 (2020) e03258 Figure 2. Formation of DNA polymerase-η reverse (Pol-η) reverse transcriptase (RT)-priming sites at activation-induced deaminase (AID) lesions on the transcribed stand (TS) at stalled transcription bubbles initiating target site reverse transcription (TSRT). A CAG repeat within a transcription bubble is shown with tandem WGCW motifs that allow the hypothesized AID targeted deamination events. This illustrates the key AID-mediated C-to-U deamination lesion, and somatic hypermutation (SHM)-initiating step discussed in the text and in step A. in Figure 1, leading to the formation of the Pol-η RT priming step (step B. in Figure 1). This step is well documented in the immunoglobulin (Ig) SHM literature, see references in Franklin et al., (2004) and other reviews (Di Noia and Neuberger, 2007; Teng and Papavasiliou, 2007; Maul and Gearhart, 2010; Steele, 2016). A recent paper by the Gearhart group demonstrates further the generation of such single stranded nicks with 30 -OH ends (Zanotti et al., 2019). The focus here, and in Figure 1, is on how the TS is deaminated as it is this TS lesion which sets up the sequelae of downstream steps (Figure 1) that lead to the key diagnostic strand biases in Ig SHM data sets discussed in the text here, namely mutations at A sites exceed mutations at T sites (A >> T) and, in the same data sets, mutations at G sites exceed mutations at C sites (G >> C). These A-site mutations are triggered primarily by the ADAR1 mediated A-to-I modifications in the nascent pre-mRNA (Steele et al., 2006). Both these counter intuitive strand biases at A:T and G:C base pairs occur in all Ig SHM data sets (Steele, 2009) and in all off-target non-Ig data sets analysed (e.g. TP53 substrates, in numerous different cancers, Lindley and Steele, 2013). They cannot be explained by utilization of the alternative templates for DNA repair synthesis, namely the non-transcribed strand (NTS) as copying templates, thus the focus here on the TS rather than the NTS. These alternatives, particularly in relation to Pol-η-mediated DNA repair, are discussed in detail in Steele et al., (2006) and Steele (2016). It is certainly accepted that alternative explanations exist for transcription coupled repair (TCR) strand-biases in somatic mutations generated by bulky adducts (Denissenko et al., 1996, 1998), which lead to Nucleotide Excision Repair (NER)-TCR and preferential repair of the TS as discussed in detail in Figure 4. For the minor base alterations considered here, simple copying errors at AID lesions by RNA polymerase II misincorporation come into play (Kuraoka et al., 2003); certainly low level 8oxoG generated lesions by reactive oxygen species (ROS) do not result in NER-TCR strand biases on DNA repair (Thorslund et al., 2002). This may not be the case for significant clusters of 8oxoG lesions at repetitive WG sites as would be the case for ROS lesions at (CAG) n tracts in expressed genes in brain cells (Figure 4 legend and references). Thus, the AID deamination of cytosines on the TS assisted by the RNA exosome (Basu et al., 2011) leads as shown to uracil in the TS which is usually removed by a uracil DNA glycosylase (i.e. UNG) which then set up the substrate for cleavage of the abasic site by an apurinic/apyrimidinic endonuclease (i.e. APE1) and generation of free 30 OH termini on the TS. These 30 OH ends, on annealing to the newly synthesised pre-mRNA at this transcribed genomic site, create the hypothesised Pol-η RT-priming complex as shown (and step B in Figure 1). 3 A. Franklin et al. Heliyon 6 (2020) e03258 Figure 3. Reverse transcriptase mechanism of CAG repeat expansion. General schematic mechanism adapted in part from prior published papers (Panigrahi et al., 2005; Mirkin, 2007; Chan et al., 2013; Guo et al., 2016), but now using a pre-mRNA template. A. Hairpin formation (stabilized by MSH2–MSH3) and template slippage of cDNA during reverse transcription opposite pre-mRNA B. Strand invasion of duplex DNA by cDNA which anneals with the NTS to replace the TS. C. Resolution of the heteroduplex by mismatch repair. Repeat contractions could occur via endonuclease removal of the hairpin prior to reverse transcription. Note that all the post-reverse transcription steps specified in Figure 1 apply here. Thus, ‘strand invasion’ as addressed in the text and in Box 1, and in Figure 1 legend, can involve either short (~30 nt) or long (>100 nt) cDNA tracts. Strand invasion could occur via either the homologous recombination/gene conversion promoting properties of DNA polymerase-η (Kawamoto et al., 2005; McIlwraith et al., 2005) or a reverse transcription-driven RNA-templated NER-TCR process for TCR gap tracts ~30 nt (discussed in the text, Box 1, in Figure 1 legend, and outlined in detail in Figure 4). CAG, cytosine-adenine-guanine; cDNA, complementary DNA; MSH2, MutS homologue 2; MSH3, MutS homologue 3; NER, nucleotide excision repair; nt, nucleotide; NTS, non-transcribed strand; TCR, transcription-coupled repair; TS, transcribed strand. of somatic expansion delays the onset of associated pathophysiology, suggesting that it is not so much CAG copy number but perfect CAG repeat copy number that rate limits somatic expansion and pathology in Huntington's disease (Budworth et al., 2015). Nevertheless, excessively expanded repeats result in fragile sites on chromosomes (Richards, 2016) and thus have genomic neurodegenerative disease consequences in the non-dividing cells of the brain, producing aberrantly misfolded proteins and toxic RNA transcripts (Mirkin, 2007; Nalavade et al., 2013; Usdin et al., 2015). Perhaps best known are the (CAG)n or polyQ expansion diseases such as spinal bulbar muscular atrophy (SBMA), dentatorubral pallidoluysian atrophy (DRPLA), Huntington's disease (HD), and various variants of spinocerebellar ataxia (SCA1,2,3,6,7,12,17). Other diseases include: the (CTG)n expansions such as myotonic dystrophy type 1 (DM1), Huntington's disease-like 2 (HDL2) and spinocerebellar ataxia 8 (SCA8); the (CGG)n expansion diseases such as fragile X syndrome (FXS) and related expansion diseases, again also involving slippage during replication (see Table 1 in Usdin et al., 2015). Since our model applies to non-dividing cells in the brain, the emphasis here is on AID/APOBEC and ADAR deaminations coupled with transcription-linked DNA processes like target site reverse transcription rather than DNA replication. In the brain and central nervous system, there are numerous hereditary TNR expansion diseases and their somatic progression correlates (Mirkin, 2007; Usdin et al., 2015). They are often referred to as “Dynamic Mutations” (Richards, 2016) to distinguish them from more conventional and stable forms of Mendelian mutations. These diseases occur and somatically progress in severity in affected families with an earlier age of onset over successive generations. Dynamic mutations of the repeat sequence type in protein-coding regions are thus associated with molecular mechanisms responsible for non-Mendelian genetic inheritance phenomena often thought of as ‘anticipatory’. We now know from data generated using mouse models of Huntington's disease that suppression 4 A. Franklin et al. Heliyon 6 (2020) e03258 Figure 4. The reverse transcription mechanism for CAG expansions which invokes the alternative DNA polymerase-η-mediated (or DNA polymerase-κ-mediated) RNA-dependent DNA synthesis mechanism coupled to the nucleotide excision repair–transcriptioncoupled repair (NER-TCR) process for short TCR gap tracts of ~ 30 nucleotides (nt). Green, red,bluerectanglesare CAG repeat tracts. Other lines are DNA in black, cDNA in blue, and premRNA in red. A. A transcribed region over a (CAG)n tract. This is a schematic and not meant to show a 9–10 nt RNA:DNA hybrid at a transcription bubble (as in Figures 1 and 2), but to convey the idea of transcription across the tract. The gold star indicates significant DNA damage(s) to the transcribed strand (TS), and thus sensed by the RNA polymerase II elongation complex, which could be due to clustered mutagenic episodes of dysregulated enzymemediated nucleic acid deamination by AID/ APOBEC and ADAR at appropriate tandem Csite and A-site deamination motifs exposed at the RNA:DNA hybrid, on the TS itself and on the displaced non-transcribed strand (NTS) (Basu et al., 2011; Zheng et al., 2017) as discussed in Steele and Lindley (2017) or conceivably of reactive oxygen species (ROS) modifying guanines at tandem WG sites creating a significant lesion on the TS (clustered 8oxoG lesions, common at CAG repeat tracts as described in Polyzos and McMurray, 2017, and see the SBS18 WG signature in https://cancer.s anger.ac.uk/cosmic/signatures). Steps A through F summarize the potential events hypothesized (also see Box 2). Following sensing of damage on the TS, the RNA polymerase II elongation complex stalls, backtracks and allows the NER machinery of the TCR supramolecular complex (Hanawalt and Spivak, 2008; Spivak, 2016) to make 50 and 30 excisions in the TS around the lesion (A) and thus release the damaged TS region (B), exposing the single-stranded gapped section of the NTS. Unlike normal TCR where the NTS would be the template for the replicative DNA polymerases-δ or -ε, the proposed model here (C) invokes co-optionofthepre-mRNA(normalsequenceor possibly also base modified, as shown in Figure 1) as the template for gap repair reverse transcription by DNA polymerase-η or even DNA polymerase-κ (Box 2), given that the latter is known to repair such TCR gaps (Ogi and Lehmann, 2006). Slipped MSH2–MSH3-stabilized CAG repeat hairpins (as in Figure 3) form on the pre-mRNA template and complementary DNA (cDNA) synthesis continues to fill the gap (D). After RNase H activity, the reformation of the TS:NTS heteroduplex (E) then sets the stage for resolution of the nucleotide differences using the TS as the template for correction of the NTS (F). ADAR, adenosine deaminase acting on RNA; AID, activation-induced deaminase; APOBEC, apolipoprotein B mRNA editing catalytic polypeptide-like; MSH2, MutS homologue 2; MSH3, MutS homologue 3. 5 A. Franklin et al. Heliyon 6 (2020) e03258 1. This involves an interaction that sanctions the protection of beclin 1 from proteosome-mediated degradation and thus normal progression into autophagy pathways (Ashkenazi et al., 2017). syndromes, and the (GAA)n expansion diseases such as Friedreich's ataxia (FRDA). Very good and generally agreed upon summaries of the various genes that can be affected, their normal repeat lengths and the range and extent of pathogenic repeat lengths can be found in Table 1 of Usdin et al. (2015), Table 2 of Richards (2016), and also in Figure 1 of Polyzos and McMurray (2017). The current expansion mechanisms invoke MutS homologue (MSH)2–MSH3 hairpin-stabilized initiation during replication on the leading strand (Mirkin, 2007) or, the now consensus view, more generally during local DNA repair across the genome (Polyzos and McMurray, 2017). That is, as we discuss later, local error-prone DNA repair on the nascent gap-filling strand assisted via MSH2–MSH3 hairpin-stabilized configurations aiding priming initiation (Panigrahi et al., 2005; Mirkin, 2007; Polyzos and McMurray, 2017), and possibly involving DNA polymerases-β and -δ (Chan et al., 2013; Guo et al., 2016). The first molecular description of (CAG)n expansion tracts was by La Spada et al. (1991) in mutations causing X-linked SBMA of the androgen receptor gene. This initial study clearly showed that in diseased individuals, the CAG repeat tracts expand in the 50 -to-30 direction (i.e. with the same transcriptional polarity of the pre-mRNA). This was soon followed by a similar study showing CAG expansion tracts in exon 1 of the IT15 gene which encodes the huntingtin protein in HD patients (MacDonald et al., 1993). We now know that about 30 or more hereditary diseases in humans (as curated at the OMIM database) result from similar somatic expansions of codon and other longer repeats in intronic or UTR encoding genomic DNA (Mirkin, 2007; Nalavade et al., 2013; Usdin et al., 2015; Richards, 2016; Polyzos and McMurray, 2017). Such expanded DNA in transcribed regions results in physiologically ‘toxic’ structural features in both aberrantly misfolded proteins and mRNA transcripts, and are thus disruptive to basic biochemical pathways. As discussed, the repeat expansions also occur in the transcribed non-protein coding 50 and 30 flanks and intronic regions causing debilitating diseases also through abnormal folding of proteins and RNA transcripts (Mirkin, 2007; Nalavade et al., 2013; Usdin et al., 2015). 4. Dysregulated mutagenic activity of deaminases and base sequence features of (CAG)n and similar tracts AID/APOBEC-mediated C-to-U are predicted to target C residues that are in the primary repeat motif WGCW in HD and related diseases (W ¼ A or T; targeted C residue underlined). Thus, in relation to known mutagenic targeting preferences of AID/APOBEC and ADAR deaminases across diseased genomes (e.g Lindley, 2013; Lindley et al., 2016), (CAG)n and related repeats such as (CTG)n, (CGG)n, (GAA)n, as well as longer repeats such as (CCTG)n and (ATTCT)n (see Table 1 in Usdin et al., 2015) possess features that could lead to dysregulated and thus pathogenic deaminase targeting. Ig variable region exons are assembled during B cell development from variable (V), diversity (D) and joining (J) gene segments and are hence referred to generically as V(D)J sequences. WGCW motifs are enriched at V(D)J sequences and exist as tandemly arranged repeats (Di Noia and Neuberger, 2007; Teng and Papavasiliou, 2007; Steele, 2016). These tandemly arranged C-centered motifs are targeted by AID in a regulated manner at transcription bubbles at rearranged V(D)J genes to initiate Ig SHM in B cells (Maul and Gearhart, 2010; Heltzel and Gearhart, 2019). They are also enriched at switch regions as part of the transcription-dependent R-loop formation during Ig class switch recombination (Yu and Lieber, 2003; Maul and Gearhart, 2010; Heltzel and Gearhart, 2019). Thus, during antigen-stimulated Ig SHM episodes at V(D)J loci in germinal center B cells in vivo, multiple nearby WGCW motifs, often overlapping and concentrated in the sequences termed complementarity determining regions which encode epitope contact residues, can be C-to-U-deaminated by AID causing staggered nicks (after base excision repair generating abasic sites and APE1 endonuclease action, Figure 2) and potentially double-strand breaks (DSBs). These DSBs are ideal targets for DNA repair via homologous recombination (Jasin and Rothstein, 2013) and gene conversion processes, both of which are able to be stimulated by DNA polymerase-η (McIlwraith et al., 2005; Kawamoto et al., 2005, Box 1). Such DSB lesions can also be the target of RNA-templated DNA repair in yeast model systems mediated presumably by replicative DNA polymerase-α and -δ (Storici et al., 2007, Box 2). This is an important point in regard to the mechanism shown in Figure 1 as DNA polymerase-η is the sole error-prone DNA polymerase known to be involved in physiological Ig SHM in vivo (Delbos et al., 2007). More information around the RT activity of human DNA polymerase-η is outlined in Box 2. The tandem CAG, CTG and CGG repeats in coding regions or UTRs in many protein-coding genes (targeted C residues underlined) will thus provide rich targets for cytosine deamination if the AID/APOBEC deamination events become dysregulated and go ‘off target’ as appears to occur across the genome in cancer (Lindley, 2013; Lindley and Steele, 2013; Lindley et al., 2016). Thus, in this context, the polyP-encoding tract in exon 1 of the Huntington's gene (IT15), just downstream of the polyQ-encoding tract, also provides an ideal deamination substrate for other cytidine deaminases known to have target substrates that overlap those of AID. For example, CCN motifs accessible in single-stranded DNA (ssDNA) substrates, such as occurs during displacement of the non-transcribed strand (NTS) at translocating transcription bubbles (Figure 1), could serve as substrates for editing by APOBEC3G (Beale et al., 2004). In this regard the reverse complement of the common 50 UTR repeat nucleotide triplet CGG in FXS and related syndromes (i.e. CCG) is a common APOBEC3G motif and would represent a potential tandem array deamination target in the DNA of an expressed gene. In the same vein, the intronic (ATTCT)n repeat (as seen in spinocerebellar ataxia 10 [SCA10]) and the (GAA)n expansion intronic repeats (as seen in FRDA) present as possible transcription-linked ADAR1 motifs in both nascent double-stranded (dsRNA) hairpins and RNA:DNA hybrids 3. Function of polyQ and related TNR homopolymer tracts in health and disease PolyQ and similar repeat codon tracts occur in most proteins (Oma et al., 2004; Willadsen et al., 2013), presumably assisting normal interaction with other proteins, intracellular localisation and supramolecular complexes (Oma et al., 2004; Willadsen et al., 2013; Huttlin et al., 2017). In healthy individuals, the lengths for the huntingtin gene (CAG)n repeats range from n ~ 10–30, yet when they expand to n >37 repeats, overt and progressive HD symptoms become apparent (La Spada et al., 1991; MacDonald et al., 1993; Mirkin, 2007; Nalavade et al., 2013; Usdin et al., 2015; Richards, 2016; Polyzos and McMurray, 2017). Thus, homopolymer tracts are a normal feature of most proteins. Across representative animal and plant species examined, many normal (wild-type) cytoplasmic proteins have functional polyQ tracts (Willadsen et al., 2013). An analysis of six different species including Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus and Homo sapiens identified the number of TNR tracts in proteins as 247, 1,947, 559, 3,996, 79,727 and 35,736, respectively (Willadsen et al., 2013). This implies that the great majority of protein-coding genes have embedded TNR tracts serving interactome network functions (Huttlin et al., 2017) involving ‘supramolecular protein-nucleic acid complexes’ such as transcription factor ‘mosaic’ assemblies in promoter regions, as well as complex molecular machines such as ribosomes, spliceosomes, RNA polymerase II elongation complexes and the DNA replication apparatus. PolyQ and similar amino acid repeat tracts also support a range of protein-protein interactions necessary for the dynamic processing of autologous proteins. For example, a protein widely expressed in the brain is ataxin, a deubiquitinating enzyme. Its polyQ domain in wild-type ataxin-3 enables it to interact with the key autophagy initiator beclin 6 A. Franklin et al. Heliyon 6 (2020) e03258 Box 1 How does the cDNA invade to form a heteroduplex? This explanation relates to steps B–E in Figure 1. How does the cDNA corresponding to the TS invade to form the first heteroduplex with the NTS (steps C and D in Figure 1)? Step B shows the predicted, and necessary, DNA polymerase-η RT-priming complex of nicked TS DNA with a 30 OH end annealed to the pre-mRNA. In previous iterations of the RT model (Franklin et al., 2004), the strand invasion step has been assumed to occur once the target site reverse transcription step is initiated as in Luan et al. (1993). The cDNA is initiated and is contiguous with the TS on the 50 side. There are at least two types of ‘strand invasion’ mechanisms following RNaseH removal of the pre-mRNA from the RNA:cDNA heteroduplex (Step C). For long cDNA tracts (>100 nt) we assume that the known homologous recombination/gene conversion promoting properties of human DNA polymerase-η allow this to happen (again after RNase H removes the annealed template pre-mRNA), as in the canonical immunoglobulin variable region gene conversion system in chicken B cells (Kawamoto et al., 2005; McIlwraith et al., 2005). For shorter tracts of up to 30 nt, which is the approximate normal maximum tract length of most CAG repeats in the known genes subject to repeat expansion (Usdin et al., 2015; Richards, 2016), it is expected that the NER-TCR machinery will act to generate excisions on both the 50 and 30 side of the damaged TS around the stalled transcription bubble, exposing an ~ 30 nt gap (Bowman et al., 1997). Normal TCR-directed gap filling (see Fig. 2 in Hanawalt and Spivak, 2008; Spivak, 2016) is now templated for cDNA synthesis of the TS opposite the pre-mRNA as shown in steps B and C, followed by ligation on the 30 side of the gap as heteroduplex is formed (Step D). In further support of the TCR alternative for ‘strand invasion’ is the fact that the gap filling to re-synthesize the ~30 nt TS gap, which equates to the excised section in normal TCR damage responses (Hanawalt and Spivak, 2008; Spivak, 2016), is a step that involves DNA polymerases-δ, -ε and/or Y-family DNA polymerase-κ (see Ogi and Lehmann, 2006). This is important in relation to the RT model for somatic hypermutation outlined in Figure 1 (and for potential CAG expansions as shown in Figure 3) because three of the human Y-family polymerases (i.e. DNA polymerases-η, -ι and -κ) can perform RNA-dependent DNA synthesis, as previously shown by Franklin et al. (2004) and see Box 2. This now adds to the likelihood that a RT-driven RNA-templated NER-TCR process may happen in normal cases of translesion bypass repair. Indeed RNA-templated DNA repair of double strand breaks has also been demonstrated in S. cerevisiae by Storici et al., (2007) (e.g. involving yeast replicative DNA polymerases-α and -δ; see comparable primer extension data of  5–10 nt opposite RNA templates in Fig. 3 in that paper). In summary, the cDNA strand invasion steps for C through D in Figure 1 can, from available evidence, be executed for long cDNA tracts by the established homologous recombination/gene conversion promoting properties of human DNA polymerase-η (for tracts >100 nt), or via a DNA polymerase-η or DNA polymerase-κ RT-driven RNA-templated NER-TCR process for TCR gap tracts of ~30 nt. The potential steps of the second alternative for CAG expansions are outlined in Figure 3 in some detail. cDNA, complementary DNA; NER, nucleotide excision repair; nt, nucleotide; NTS, non-transcribed strand; TS, transcribed strand; RT, reverse transcriptase; TCR, transcription-coupled repair. heteroduplex DNA. As discussed in the legend to Figure 1 and Box 1, this ‘strand invasion’ process could be of limited tract length (30 nucleotides) by co-option of the 50 and 30 nicks on the TS by the complex responsible for the normal transcription-coupled repair (TCR) pathway of nucleotide excision repair (NER) known as NER-TCR (see Figure 2 in Hanawalt and Spivak, 2008; Spivak, 2016). This would create a ~30 nt gap (Bowman et al., 1997) that can be filled in by DNA polymerase-η performing reverse transcription extending from the nicked 30 –OH–primed TS DNA. Alternatively, the 30 -OH TS priming site can be generated by AID-mediated deamination at the WGCW sites in the repeat (CAG)n or (CTG)n tracts. The length of such cDNA tracts synthesized by DNA polymerase-η can be promoted by the homologous recombination (gene conversion) properties that are also associated with its activity. In the chicken Ig gene conversion system, DNA polymerase-η deficiency causes a significant decrease in the frequency of gene conversion, with increased tract lengths in residual gene conversion events (Kawamoto et al., 2005). These results are compatible with other biochemical studies on homologous recombination promoting properties of human DNA polymerase-η (McIlwraith et al., 2005). As also addressed in Box 1, the results can be understood as DNA polymerase-η promoting DNA synthesis from strand invasion intermediates of homologous recombination, thus allowing invasion of the target V(D)J sequence by a pseudo V gene donor in the case of chicken gene conversion to generate a new Ig variable region-encoding tract (which can be up to several 100 nucleotides in length, in contrast to the ~30 nucleotide gap synthesis tract in conventional NER-TCR just discussed). A D-loop is a DNA structure in which the duplex DNA is separated (unwound) and then held apart by a third homologous DNA strand in a triplex structure. In other triplex RNA-DNA structures, weaker non-Watson-Crick base pairs form Hoogsteen base pairing in which the third strand can be either in parallel or in the reverse orientation (see Li et al., 2016; Buske et al., 2012). In these structures, the triplex base hydrogen bonding involves non-Watson-Crick Hoogsteen hydrogen bonding such that A can pair with A, G with G and so on, pairings which are far weaker in strength but allow specific sequence identification (Zheng et al., 2017; Steele and Lindley, 2017). The RNA:DNA hybrid target substrates are similar to the numbers of WA-rich targets both within the intronic Alu repeat elements themselves (the well known WA rich sequences in the Alu central region) and their inverted, and spaced, Alu snap-back derivatives, that are known to constitute the main targets of ADAR-mediated A-to-I RNA editing in vivo in the brain (Paz-Yaacov et al., 2010; Picardi et al., 2015). Returning to the cancer analogy and the link with deaminase action, as with the triggering of Ig SHM itself (reviewed in Di Noia and Neuberger, 2007, Teng and Papavasiliou, 2007, and again in Steele, 2009, Maul and Gearhart, 2010, and Steele, 2016), we and others have analysed and interpreted cancer data which suggest that DNA C-to-U deamination at off-target (i.e. non-Ig) genomic sites by AID/APOBEC deaminases triggers the recruitment of an ‘Ig SHM-like’ response (Lindley, 2013; Lindley and Steele, 2013). Many non-Ig protein-coding exons exist with (CWG)n repeat loci rich in clustered WGCW motifs (Willadsen et al., 2013). We also call these deamination events at off-target sites ‘dysregulated Ig SHM-like responses’ (Steele and Lindley, 2010; Lindley, 2013; Lindley and Steele, 2013; Lindley et al., 2016). We now know from the recent TCGA analyses of Niavarani et al. (2018) that TNR expansions are a very common feature in pan-cancer genome exomic data, accounting for 1–2% of DNA sequence modifications in cancer genomes, a similar frequency to the in-frame codon repeats observed in human Ig SHM data, as reported by Wilson et al. (1998a,b), and Reason and Zhou (2006). Thus, dysregulated in-frame expansions while rare are frequent enough outside the brain to be detected during SHM itself and in progressing somatic diseases such as cancer. We propose that for an expansion event to be potentiated during complementary DNA (cDNA) synthesis, facilitated by DNA polymerase-η extension of the slipped MSH2–MSH3-stabilized CAG hairpin in the transcribed strand (TS) generated via reverse transcription (Figure 3), the premRNA target site reverse transcription (TSRT) process (Luan et al., 1993) must allow the cDNA to invade the site (Box 1) to create new mutated 7 A. Franklin et al. Heliyon 6 (2020) e03258 Box 2 Reverse transcriptase activity of DNA Polymerase-η and DNA Polymerase-κ During the peer review of this article, the basic validity of the reverse transcriptase (RT) model of immunoglobulin somatic hypermutation driven by the RNA-dependent DNA synthesis activity of human DNA polymerase-η was queried, mainly because of our inappropriate citation of key papers by the Su et al. group. We should have cited both Su et al., (2017) and Su et al., (2019) rather than just the more recent 2019 paper of the group. This confusion nevertheless generated this useful clarifying Glossary Box which now addresses this sceptical viewpoint which we believe is reasonably widespread in the molecular immunology community (at least). The in vitro observations in Franklin et al. (2004) on human Y-family DNA translesion DNA polymerases-η, -κ, -ι were made using an indirect, PCR-based detection method, a product enhanced real time (PERT) PCR assay. Moreover, although Su et al. (2017, 2019) recently demonstrated independently the RT activity of human DNA polymerase-η, the efficiency of polymerization that was reported in the Su et al., (2019) paper, in contrast to that in Su et al., (2017), was very low opposite RNA template relative to DNA template, with no primer extension being observed beyond incorporation of only a single nucleotide. So just reading Su et al., (2019) by itself can create the misleading impression that human DNA polymerase-η is a poor cellular RT that is hardly likely to support the continuous insertion of dozens of nucleotides as we have proposed in this paper (and see Blanden et al., 2004). So we have now critically re-read the key papers on this issue by Su et al. (2017, 2019), particularly the primer extension assays used in the earlier article (Su et al., 2017). These workers annealed short oligonucleotide sequences in vitro to create DNA/RNA substrates. The primer extension data in these in vitro biochemical primer extension assays with purified enzymes completely confirm our prior work and conclusions using the PERT assay (Franklin et al., 2004). Our work showed that elongation of complementary DNA (cDNA) copies opposite the MS2 phage RNA template annealed to a DNA primer yielded extension products of at least 27–37 nucleotides. The critical confirmatory data is presented in Figure 4 in the report by Su et al. (2017), which shows DNA primer extension data opposite RNA template. Specifically, a 50 -to-30 DNA primer was annealed to a longer 30 -to-50 RNA template (longer by 5 nucleotides); the maximum possible product size generated by cDNA extension of the DNA primer that could be detectable by polyacrylamide gel analysis was 5 nucleotides. These data compare favourably with the minimum 27–37 nucleotide cDNA products within 1 h incubation detected in the PERT assay of Franklin et al. (2004). So the primer extension data of Su et al., 2017 provide a clear confirmatory demonstration of genuine reverse transcription not only by human DNA polymerase-η but also to a lesser extent by human DNA polymerase-κ for purified enzymes in vitro as in Franklin et al. (2004). The relative RT efficiencies reported are also similar to those reported in Franklin et al. (2004). Thus the comparison with the more efficient HIV-1 RT is very informative and also similar to the relative comparisons in Franklin et al. (2004). But the additional enzyme kinetic information reported in Su et al., (2017) (in their Table 1) is new quantitative data and very important in the understanding of the relative efficiency of the RT activity of human DNA polymerase-η. The insertion of dC opposite template rG (as a measure of catalytic efficiency) for both human DNA polymerase-η and HIV-1 RT is very similar, allowing them to conclude that human DNA polymerase-η is a relatively efficient cellular RT. While similar results in principle to human DNA polymerase-η were observed for human DNA polymerase-κ, the latter polymerase was clearly less efficient as compared to the former in terms of RNA-dependent DNA synthesis activity (Su et al., 2017). It is important to qualify that all of these biochemical data were gathered under conditions in vitro using purified polymerases. These conditions are clearly far removed from in vivo physiological conditions in living cells which in most well studied cases involve supramolecular complexes and regulated interactions of many functional proteins among themselves and with nucleic acid molecules. Thus, in vivo, we should expect the replication clamp proliferating cell nuclear antigen (PCNA) with the single strand stabilizing proteins replication protein A (RPA) with replication factor C (RFC) to participate in improving and regulating the processivity of the DNA polymerases, particularly translesion human DNA polymerase-η (Haracska et al., 2001). Indeed we conducted in vitro experiments involving PCNA, RPA and RFC and found that human DNA polymerase-η activity in the PERT assay was enhanced at least four-fold by the addition of these proteins (Franklin, 2004), a result which implies that under in vivo conditions in living cells, processive cDNA synthesis can be expected. Long tract cDNA synthesis in vivo via the RT activity of human DNA polymerase-η is expected at rearranged immunoglobulin loci (Blanden et al., 2004) and is expected when the 5’ boundaries of the distribution of somatic mutations are critically evaluated (Blanden et al., 2004). We note that Krijger et al. (2011) have shown ubiquitination-independent PCNA activation of DNA polymerase-η during physiological in vivo somatic hypermutation and DNA damage tolerance in a murine system. The follow up work reported in Su et al. (2019) uses human cell extracts. In these experiments (see Figure 4 in Su et al., 2019), the “presence of RNA strands in the annealed DNA primer/RNA template complex caused the substrates to be degraded more easily than with the DNA/DNA substrate.” They speculate that this extensive degradation is probably caused by RNase H1 and RNase H2 in the cell extracts. So it is actually a race in these experiments to extend the cDNA product by 5 nucleotides before degradation of the substrate complex (or extension products themselves). Nevertheless, the authors report clear human DNA polymerase-η-dependent cDNA extensions of one nucleotide opposite the RNA template, which allows the authors to conclude that in these ‘cellular environments’ the results demonstrate the critical role of human DNA polymerase-η in reverse transcription and indicate that human DNA polymerase-η is a key reverse transcriptase in human cells (Su et al., 2019), thus extending their work on purified polymerases in primer extension assays in vitro (Su et al., 2017). 8 A. Franklin et al. Heliyon 6 (2020) e03258 non-ATG-mediated translated protein products, which can be very toxic to the cell (Pearson et al., 1997; Cleary et al., 2018). Thus, the question again is: How are such (CAG)n and related repeats expanded in pre-mRNA in the absence of cell division? We propose that the error-prone RT mechanism for Ig SHM (Figures 1 and 2, Box 1, Box 2) has the potential to be adapted as an explanation for (CAG)n and related expansion diseases, thereby accounting for the generation of variant premRNA expanded repeats (Figures 3 and 4), as well as potentially mutated sequences (i.e. somatic point mutations) in the flanking regions of TNR expansions. These new pre-mRNA sequences are then cDNA copied and locked back into the chromosomal DNA at that site (Luan et al., 1993) via the targeted RT action of DNA polymerase-η (or DNA polymerase-κ, Figure 4). These potential repeat expansion steps can also be adapted to the normal physiological Ig SHM process where about 1–2% of variant sequences in a hypermutated set of somatically mutated derivatives of rearranged V(D)J regions contain short 1–3 nucleotide indel repeats, including the previously discussed (Steele, 2016) in-frame codon expansions and contractions (Wilson et al., 1998a, 1998b; Reason and Zhou, 2006). Moreover, the proposed mechanism can also potentially lead to repeat contractions through hairpin removal by local DNA repair prior to reverse transcription. If these processes take place in the transcribed regions of expressed genes in germ cells, similar consequences for polymorphism generation are possible if off-target ‘Ig SHM-like’ responses are so activated. We therefore expect the TNR expansion mechanism proposed here to be a general mechanism occurring across the genome for at least the (CAG)n, (CTG)n and (CGG)n diseases, as well as being a feature of other progressive somatic diseases such as cancer (see Dai and Wong, 2003 regarding breast cancer, and Niavarani et al., 2018 regarding many other cancers). Cancer genomes are known to display the mutation signature of AID/APOBEC off-target Ig SHM-like responses (Lindley, 2013; Lindley and Steele, 2013; Lindley et al., 2016). Further, the varied secondary RNA fold-back dsRNA structural conformations produced from TNR loci that are mutated are likely to be functionally altered, particularly via ADAR1-mediated RNA and DNA editing in RNA:DNA hybrids at transcription bubbles (Steele and Lindley, 2017) and also at A/C mismatches themselves in post-transcriptional snapback dsRNA structures in both exons and in 50 and 3’ UTRs. Such A-to-I alterations could thus disrupt evolutionarily conserved RNA secondary structures in regulatory long non-coding RNAs (Smith et al., 2013, 2017). over a longer region. An example of this type of triplex sequence matching can be found in Buske et al. (2011, 2012) for AG-rich enhancer/promoter regions. 5. Questions in advance Given the expanded range of opportunities that we are now aware of for deaminases to target substrates in repeat tracts, it has been useful for us to pose the following three questions:  What are the likely mechanisms of (CAGn)/polyQ and related TNR expansion diseases?  Do these mechanisms incorporate all of the known molecular processes associated with expandable repeat diseases in vivo?  How do repeat expansion diseases occur in the absence of cell division? Answers to these questions are of special relevance to familial brain expansion diseases (Mirkin, 2007; Usdin et al., 2015; Richards, 2016; Polyzos and McMurray, 2017) and their idiopathic relatives (Bozza et al., 1995; Ishikawa et al., 1999; Kim et al., 2007). Here, we propose an augmented molecular explanation that implicates the involvement of an alternate template and an alternate DNA repair polymerase (both of which occur in the absence of DNA replication) in known localized DNA-based repair mechanisms. Fundamental to our expanded view we ask: Is there a plausible role for pre-mRNA template intermediates and target-site reverse transcription involving DNA polymerase-η (as implicated for Ig SHM per se at rearranged V(D)J genes as shown in Figure 1) in TNR and related repeat expansion diseases occurring in transcribed regions of the genome (Figure 3)? Another unanswered question is why such diseases are particularly prominent in brain and neuronal cells? For this, we have no specific answer. A general answer could involve the concept of the ‘inflamed brain’ and prion misfolding diseases as recognized now in Alzheimer's disease (Jaunmuktane et al., 2015; McCaulley and Grush, 2017). This concept is gathering momentum, both in scientific circles and in the clinic. It has much validity as it is known that chronic inflammatory diseases in the periphery, via immune cytokine cascades, can functionally communicate across the blood–brain barrier and activate enhanced “innate immunity states” in the microglial cells of the brain with untoward dysregulated consequences for normal brain function (Bullmore, 2018). Thus, tissue-localized aberrations in molecular innate immunity through off-target AID/APOBEC activation and targeting of C residues within deamination motifs (the prominent WGCW motifs in CAG repeats) might as a consequence precipitate aberrant Ig SHM responses in brain tissues. Disease triggers, in combination or in part, might include surgical central nervous system injuries associated with spinal taps (Gal-Mark et al., 2017), chronic peripheral inflammatory diseases (Bullmore, 2018), head knock brain injuries, pathogen infections and autoimmune inflammatory infiltrations in conjunction with a leaky blood–brain barrier (Myslinski, 2014; Montagne et al., 2015), or triggering of intrinsic endogenous dysregulated innate immune responses by snap-back dsRNA TNRs themselves (Richards et al., 2018; van Eyk et al., 2019). Activation of APOBEC and ADAR deaminases are known consequences of interferon-dependent innate immune response cascades (Schoggins and Rice, 2011; Schneider et al., 2014). In healthy brain tissues, expression of AID/APOBEC deaminases are low to undetectable (Refsland et al., 2010; Koning et al., 2009), while variable expression of ADAR isoforms are a normal physiological feature of the healthy brain (Picardi et al., 2015). Finally, we ask, how do potentially expandable pathogenic TNR and related tracts arise in non-protein-coding introns as well as 50 and 3’ untranslated regions (Mirkin, 2007; Usdin et al., 2015; Polyzos and McMurray, 2017)? One possibility is that they have been dispersed there in the evolutionary past as a consequence of retrotransposition events of fragments of RNA transcripts from coding regions. These transcribed yet non-protein-coding repeats often predispose to initiation of harmful 6. What is the mechanism of CAG repeat expansions in nondividing cells? Most models of TNR expansion depend on hairpin-stabilized slippage and are based on DNA replication and repair models (Panigrahi et al., 2005; Mirkin, 2007; Chan et al., 2013 Guo et al., 2016), although local DNA repair models are being increasingly considered (reviewed in Polyzos and McMurray, 2017). Indeed, MSH2-associated mismatch repair deficiency actually leads to an absence of CAG repeat expansion, indicating that CAG repeat expansion requires an intact mismatch repair system (Manley et al., 1999). Hairpins in the leading strand, if stabilized (by MSH2–MSH3), can generate stable slippage events opposite the template DNA strand allowing priming of further leading strand DNA synthesis. After this, the relaxation of the retained hairpin leads to expansion of the number of TNR at the site of CAG repeats and other TNRs in the leading strand. In our view, there are two possible molecular explanations for local DNA repair mechanisms based on stabilized hairpin slippage priming: Mechanism 1. The first is based on DNA replication of the leading strand or localized DNA repair (Mirkin, 2007). This is a repair pathway involves DNA polymerase-β (assisted by DNA polymerase-δ) recruited by MSH2–MSH3 heterodimers targeting CAG repeat hairpins and resulting in stable slipped structures for priming of synthesis of the leading DNA strand (as described by Chan et al., 2013, Guo et al., 2016, and as extensively further reviewed by Polyzos and McMurray, 2017). This 9 A. Franklin et al. Heliyon 6 (2020) e03258 the NTS at the first position of the codon would result in the creation of ‘TAG’ stop codons thus resulting in N-terminal truncated proteins which are likely to never be recovered in DNA sequence collections (owing to purifying selection and thus censorship through nonsense-mediated decay and apoptotic cell deletion). But a G-to-A change in the third position is common. This strongly suggests C-to-U editing of the complementary DNA strand (i.e. the TS), resulting in a G-to-A mutation being incorporated into the synthesized pre-mRNA strand (as shown in Figure 1). If this deamination event is the result of an AID-mediated deamination, then it may also activate further off-target Ig SHM-like responses at polyCAG and similar tracts. Some further information and references supporting the RNA/RT mechanism proposed here are included in Figures 1, 2, 3, and 4, as well as Boxes 1 and 2. The entire spectrum of somatic point mutations known to be associated with off-target Ig SHM-like activity occur at and around the initial uracil lesion that results from C-to-U editing in DNA (Figure 2). With respect to the NTS (by convention), in vivo data also show that mutations at A residues exceed mutations at T residues (A >> T) and mutations at G residues exceed mutations at C residues (G >> C) as expected by the RNA/ RT mechanism given that the great majority of base substitutions and base modifications accrue in the pre-mRNA, as highlighted in Figure 1 and discussed in Figure 2 legend (Steele, 2009; Lindley and Steele, 2013). This is expanded on in the Supplementary Information File. The deaminase-driven RT off-target Ig SHM model outlined here is consistent with the recent data reportedby Su and Freudenreich (2017). These investigators showed that a dependence on C residue deamination-coupled BER resulted in CAG repeat fragility and instability in Saccharomyces cerevisiae. It is also consistent with the data reported in Neil et al. (2018) on RNA:DNA hybrid instabilities promoting GAA repeat expansions in FRDA via break-induced DNA replication. These reports are also consistent with our proposal that CAG and GAA repeat fragility and expansion data fit within the aegis of the RT Ig SHM model, albeit now acting in a dysregulated and off-target manner across the genome and guided by pre-mRNA templates around which all the mutagenic action depends (Figures 1, 3, and 4). model can explain (CAG)n expansions in the DNA of non-dividing cells via conventional localized DNA repair concepts. We also note that, in yeast model systems of FRDA, RNA:DNA hybrid instabilities have been explained via transcription-generated RNA:DNA hybrids promoting (GAA)n repeat expansions in FRDA via break-induced DNA replication (Neil et al., 2018). Both of these mechanisms, however, can be classified as DNA-based. Mechanism 2. The second possible explanation is based on mutagenic polynucleotide copying sequelae at transcription bubbles (Figure 1) now applied to CAG expansion diseases (Figures 3 and 4). This implies an error-prone RNA/RT-based DNA repair pathway, triggered and involving an ‘off-target Ig SHM-like response’. This invokes: a. specific codon-context targeting of cytosines by AID/APOBEC deaminases to unpaired C residues in ssDNA regions of both the displaced NTS and the TS during transcription (see Figure 1); b. translesion Y-family DNA polymerase-η synthesizing DNA opposite RNA (Franklin et al., 2004, Su Y et al., 2017, Su Y et al., 2019, Box 2), and; c. ADAR1 targeting at and around transcription bubbles via binding to negatively supercoiled Z-DNA (Steele et al., 2006). This RT model of Ig SHM has also been advanced to explain off-target (non-Ig) somatic mutagenesis across the progressing cancer genome (Steele and Lindley 2010, 2017; Lindley, 2013; Lindley and Steele, 2013; Lindley et al., 2016). The off-target Ig SHM-like model, based on aberrant DNA repair and reverse transcription, is assumed to involve recruitment of DNA polymerase-η via MSH2–MSH3/MSH2–MSH6 heterodimers engaging GU mispairs and short bulges/hairpins (compare Wilson et al., 2005). Shown in Figures 3 and 4, this model also provides a plausible explanation for such (CAG)n expansions in non-dividing cells, but also now involving the RT activity of an additional Y-family member, namely DNA polymerase-κ (Franklin et al., 2004). This could be triggered by an innate immune response to localized inflammation that activates off-target AID/APOBEC-mediated deamination of C residues in the canonical WGCW motifs in the NTS and the TS during transcription (Steele and Lindley, 2017). The second explanation (Figures 3 and 4) is identical in every way to the first DNA-based model, except that the copying template is now the homologous sequence embodied in the pre-mRNA. Additionally, the DNA repair enzyme is the Y-family translesion DNA polymerase-η (and potentially DNA polymerase-κ as well), which performs reverse transcription to synthesize an error-prone cDNA copy of the TS, a downstream process after passage of the transcription bubble through that transcribed region. In our view, the second explanation provides a plausible sequelae of molecular steps to explain (CAG)n expansions at RNA polymerase II-transcribed regions in both dividing and non-dividing cells. Is the signature of C-to-U deamination mediated by the AID/APOBEC enzymes evident in (CAG)n expansion data? In all (CAG)n expansion sequence collections examined, there are (CAG)n tracts interspersed with CAA codons (also coding for glutamine). So, these third position silent mutations do not change the protein sequence. However, as Nalavade et al. (2013) point out: 7. Direct evidence for DNA polymerase-η involvement in (CAG)n expansions? Reviewers have made us aware of the work of Dixon and Lahue (2002) in a yeast model system. (CAG)n contraction and expansion in this system is essentially unaffected by a deficiency in DNA polymerase-η (Rad30), suggesting that it has little or no effect on either CAG contraction or expansion. We note, however, that one of the three contraction trials reported by Dixon and Lahue (2002) showed a five-fold reduction in (CAG)n contractions in the absence of DNA polymerase-η (while no effects on expansions were noted in other experiments). Several points can be made about these data. Yeast model systems emphasize replicative (CAG)n expansions (or contractions). A number of polymerases acting in concert with MSH2–MSH3 heterodimers could be involved. Apart from the two investigated yeast translesion DNA polymerases-η and -ζ as such (and thus potentially assisting (CAG)n expansions/contractions), there is the potential RNA-templated DNA repair activity of yeast DNA polymerases-α and -δ (Storici et al., 2007). It is therefore not surprising to us that there is little effect of DNA polymerase-η ablation in such a system. Our focus here is exclusively on RNA polymerase II-transcribed regions in non-dividing human cells (the situation that would be expected to apply in the brain). In these type of situations, off-target or dysregulated AID/APOBEC (and ADAR) deaminases can potentially target C residues (and A residues) in substrates in the context of stalled transcription bubbles at RNA:DNA hybrids (Steele and Lindley, 2017). In a normal length 10–30 nucleotide CAG repeat tract (Usdin et al., 2015; Richards, 2016) we can expect that the tandem array of AID motifs (i.e. WGCW) will present many potential deamination targets. Many WGCW motifs could be deaminated in a dysregulated ‘hypermutation’ episode inducing a DNA damage response to the affected CAG repeat tract. Indeed, prior DNA duplex damage by reactive oxygen species and 8oxoG generation “Silent mutations in CAG repeats can also lead to disease, such as SCA2 (caused by CAG repeats in ataxin 2 [ATXN2]), wherein the CAA codons normally interspersed within the CAG repeat are absent in patients, leading to an enhanced uninterrupted CAG repeat. As CAG and CAA both code for Q, there are no resulting amino-acid changes, indicating that mRNA level changes are sufficient for disease development. There are significant structural consequences of CAA interruptions on the hairpin formations...” We quote this in full because it touches on key aspects of our expanded molecular explanation. Also, it underlines the fact that ‘toxic’ RNA structures can be the result of TRN expansions in transcribed regions. We expect C-to-U deamination signatures on both the NTS and the TS at transcription bubbles as well (Figure 1). A direct C-to-U change in 10 A. Franklin et al. Heliyon 6 (2020) e03258 8. Frequency of such RT-driven expansion events in vivo? can also be expected as a common DNA damage event at (CNG)n tracts (Polyzos and McMurray, 2017, Figure 4). If an innate immune response initiates a deaminase-driven ‘hypermutation’ cascade, this could potentially mutate several of the numerous WGCW tandem sites in the normal 10–30 nucleotide repeat (AID/APOBEC enzymes tend to act in a processive manner on DNA substrates, Chelico et al., 2009), and the RNA polymerase II complex will certainly sense this type of DNA damage in advance and organize to backtrack and recruit the NER-TCR repair machinery (Hanawalt and Spivak, 2008; Spivak, 2016). The density of WGCW motifs in CAG repeat tracts that could act as deamination targets of AID is similar to the high concentrations of the same or similar AID motifs in rearranged V(D)J genes and the proximal intergenic regions (see these features in the in vivo hypermutated VκOxJκ5 transgene sequence described in Steele et al., 2006 and the VκOxJκ5 and downstream Jκ5-Cκ sequence presented in the supplementary data of Steele, 2016). A reviewer has also proposed that the frequency of the proposed RT expansion events (Figures 3 and 4) is likely to be low, compared to the frequency of TNR expansions observed in model systems where instability thresholds are reached very frequently. Many model systems would be of the ‘replicative-type’ so the issues surrounding non-dividing cells would not necessarily apply. That said, we agree with this expectation. Furthermore, we anticipate that these hypothesized events (detailed in Figures 3 and 4 as per Figure 1), like the neuronal diseases themselves, are usually rare. But in the affected non-dividing cells of the brain they could have significant impact. Untoward ‘inflammatory’ triggering events with deaminase activities targeting WGCW motifs could be important to pathology. As discussed above, these could be external through breaches in the blood–brain barrier, or endogenous to affected Figure 5. Deaminase and reverse transcriptase-driven mutagenesis at transcription bubbles. 11 A. Franklin et al. Heliyon 6 (2020) e03258 brain cells related to ‘anti-self innate immune responses’ to the snap back dsRNA repeat hairpins themselves and involving ADAR1 (Liddicoat et al., 2015; Richards et al., 2018; Samuel, 2019; van Eyk et al., 2019). Additional information Supplementary content related to this article has been published online at https://doi.org/10.1016/j.heliyon.2020.e03258. 9. Summary References Our main deduction is that the RT model proposed for (CAG)n and similar expandable repeats draws upon the implications of what we now know about off-target Ig SHM-like mechanisms. The steps for RNA/RT (CAG)n repeat expansions shown in Figures 3 and 4 are thus plausible within the context of a RNA/RT model involving off-target deaminasedriven Ig SHM-like responses (Figure 1). Figure 5 summarizes this model with respect to the downstream sequelae of deaminase-mediated and RTcoupled mutagenesis at transcription bubbles. When this process is confined to Ig loci in the regulated environment of the hypermutating germinal center B lymphocyte, the beneficial result to immunological health is the affinity maturation of antibodies and protection against disease. However, it is clear that when dysregulated there is very great potential for somatic mutagenesis at multiple off-target genomic sites causing serious pathologies, such as suggested here for repeat expansion diseases in non-dividing cells and for cancer progression as described elsewhere. Thus, we propose here a distinct set of additional molecular concepts to provide a significantly augmented way of viewing the molecular mechanism of repeat expansion diseases, particularly (CAG)n-based diseases, appearing in the protein-coding regions of genes expressed in non-dividing cells. This view combines new additional pathways with pre-existing thought on (CAG)n repeat generation processes. The main advantages of adopting a RNA/RT-based model are: Ashkenazi, A., Bento, C.F., Ricketts, T., Vicinanza, M., Siddiqi, F., Pavel, M., et al., 2017. Polyglutamine tracts regulate beclin 1-dependent autophagy. Nature 545, 108–111. Basu, U., Meng, F.L., Keim, C., Grinstein, V., Pefanis, E., Eccleston, J., et al., 2011. The RNA Exosome targets the AID cytidine deaminase to both strands of transcribed duplex DNA substrates. Cell 144, 353–363. Beale, R.C.L., Petersen-Mahrt, S.K., Watt, I.N., Harris, R.S., Rada, C., Neuberger, M.S., 2004. Comparison of the different context-dependence of DNA deamination by APOBEC enzymes : correlation with mutation spectra in vivo. J. Mol. Biol. 337, 585–596. Blanden, R.V., Franklin, A., Steele, E.J., 2004. The boundaries of the distribution of somatic hypermutation of rearranged immunoglobulin variable genes. Immunol. Cell Biol. 82, 205–208. 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The main repeat motifs, AGCA or TGCT, conform to the known prominent AID-targeting deaminase motif (i.e. WGCW), which when deaminated (C-to-U) is known to activate locus-specific Ig SHM and class switch recombination. The existence of the interspersed CAA repeat in (CAG)n tracts is consistent with C-to-U deamination of the C-site on the complementary strand in the evolutionary past; that is, there is, in our view, a clear evolutionary remnant of the AID deamination C-to-U signature on the TS (the template for pre-mRNA synthesis) that is found in all data sets (the absence of the TAG signature is explained by purifying selection). 3. The RT activity of human Y-family DNA polymerases-η and -κ, given that both are bona fide high-rate error-prone translesion repair polymerases, feasibly contributes to both somatic mutation during disease progression as well as germline polymorphism in the CAG and related TNR repeats and surrounding sequence. 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Establishing women’s cancer care services in a fragile, conflict and violence affected ecosystem in Africa Groesbeck Preer Parham1,2,a , Kabongo Mukuta Mathieu3, Tankoy Gombo YouYou3, Michael L Hicks1,2,4,5,6,b , Ronda Henry-Tillman7,c Alex Mutombo3, Mukanya Mpalata Anaclet3, Mulumba Kapuku Sylvain3, Leeya Pinder2,8,d , Maya M Hicks9e , Louis Kanda10 and Mirielle Kanda10 , Department of Obstetrics and Gynecology, University of North Carolina at Chapel Hill, 101 Manning Dr, Chapel Hill, NC 27514, USA Department of Obstetrics and Gynecology, University Teaching Hospital – Women and Newborn Hospital, 10101 Nationalist Way, Lusaka, Zambia 3 Biamba Marie Mutombo Hospital, No. 9777, Boulevard Lumumba, Commune de Masina, Kinshasa, Democratic Republic of the Congo 4 St Mary Mercy Cancer Center, 36475 Five Mile Rd, Livonia, MI 48154, USA 5 St Joseph Mercy Oakland Cancer Center, 44405 Woodward Ave, Suite 202, Pontiac, MI 48324, USA 6 McLaren Macomb Medical Center, 1000 Harrington Blvd, Mount Clemens, MI 48043, USA 7 Winthrop P Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, 4301 West Markham St, Slot #725, Little Rock, AR 72205, USA 8 Department of Oncology, University of Washington, 1959 NE Pacific St, Seattle, WA 98195, USA 9 Howard University College of Medicine, 520 W St NW, Washington, DC 20059, USA 10 Dikembe Mutombo Foundation, 400 Interstate N Pkwy, Suite 1040, Atlanta, GA 30339, USA a https://orcid.org/0000-0001-5922-5990 b https://orcid.org/0000-0002-1819-155X c https://orcid.org/0000-0002-1782-9523 d https://orcid.org/0000-0002-8929-7810 d https://orcid.org/0000-0002-1993-3367 1 Research 2 Abstract Background: The majority of the world’s poorest women (income < $1.90/day) reside in fragile, conflict and violence (FCV)-affected countries, like the Democratic Republic of the Congo. Health services in these settings have traditionally focused on immediate relief efforts, communicable diseases and malnutrition. Recent data suggests there is need to widen the focus to include cancer, as its incidence and mortality rates are rising. Methods: Employing competency-based learning strategies, Congolese health professionals were trained to perform same-day cervical cancer screening and treatment of precancerous lesions of the cervix; same-day clinical breast examination and breast ultrasound diagnostics; surgical treatment of invasive cancers of the breast and cervix; and infusion of cytotoxic chemotherapy. Outpatient breast and cervical cancer care clinics, a chemotherapy suite and surgical theatres were outfitted with equipment and supplies. Correspondence to: Groesbeck Preer Parham Email: professorparham@gmail.com Results: Combining local and regional hands-on training seminars with wise infrastructure investments, a team of US and Zambian oncology experts successfully implemented a clinical service platform for women’s cancers in a private sector health facility in the Democratic Republic of the Congo. Publication costs for this article were supported by ecancer (UK Charity number 1176307). Conclusion: We forged a novel partnership between oncology health professionals from Africa and its Diaspora, international philanthropic organisations, a cancer medicine access initiative and an established African cancer centre to build women’s cancer services in a FVC-affected African setting. ecancer 2021, 15:1231; www.ecancer.org; DOI: https://doi.org/10.3332/ecancer.2021.1231 ecancer 2021, 15:1231 https://doi.org/10.3332/ecancer.2021.1231 Published: 13/05/2021 Received: 18/11/2020 Copyright: © the authors; licensee ecancermedicalscience. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1 Keywords: fragile, conflict and violence affected societies, Democratic Republic of the Congo, cervical cancer, breast cancer, African cancer centre, Dikembe Mutombo Two billion people live in areas plagued by violence and conflict, among whom are the majority of the global poor (income < $1.90/day) and a third of the world’s women [1, 2]. Since 1990, there have been over 1,100 recorded conflicts across 102 countries, and the numbers are increasing [3]. While the effects of war and conflict are profoundly devastating, they can extend beyond the battlefield. By destabilising the political institutions that help to ensure basic human rights and freedoms [4], war and conflict create the social conditions (extreme poverty, food shortages, lack of safety, overcrowded and unsanitary living arrangements) that result in disease and death within the countries in which they occur [5]. The prioritisation of health issues in fragile, conflict and violence (FCV)-affected societies is largely dependent on international funding [6]. Recent data suggests there is need to include cancer as a priority health concern in these environments, as its incidence and mortality rates are rising. Of particular importance are cancers of the cervix and breast, because of their heavy burden among the world’s poorest women [7]. To be effective, cancer care services for women residing in these resources-constrained settings must address the following needs: (A) health promotion messages that consider the social, cultural and religious norms that impede the acceptance of allopathic cancer care; (B) clinical infrastructures for screening, early detection, diagnosis and treatment; (C) workforce development that includes oncology specialists, ancillary health professionals and technicians and (D) access to high-quality, low-cost cancer medicines and opioids. Motivated by the need and our previous successes in establishing cancer care services for marginalised women in Africa and its Diaspora [8–13], we embarked on an initiative with the objective of developing and implementing a cervical and breast service platform in the Democratic Republic of the Congo (DRC), the largest fragile nation in Africa [14]. This report gives an overview of our approach to programme implementation in which workforce training was integrated with service delivery and clinical infrastructure development [9, 13]. Other manuscripts in this series provide a detailed and rigorous evaluation of the clinical outcomes of our activities [15–17]. Background and rationale The DRC is the second largest country on the African continent and major African continental crossroad being bordered by Rwanda, Uganda, Burundi and Tanzania to the east, Central African Republic and South Sudan to the north, Angola and Zambia to the south and Republic of Congo to the west. Formally established as a Belgian colony in 1908, the DRC gained independence from Belgium in 1960, became Zaire in 1972 and formally DRC in 1997. The population of 84 million people is now host to over 200 different ethnic groups and distinct languages with Lingala, Kingwana, Kikongo, Tshiluba and French considered the national languages [18]. The DRC is fraught with centuries of political destabilisation, armed clashes, internal wars and human rights violations. Much of the conflict has been centred around control of its vast reserves of gold, diamonds, tungsten, uranium, oil and coltan – an ore used in laptops and mobile phones [19]. The combination of conflict, economic plunder by multinational corporations and internal corruption has had a devastating effect on the living conditions of its people and stability of its public institutions, including the collapse of the healthcare system. According to the World Bank, approximately 70% of the DRC’s 84 million inhabitants survive on less than $1.90/day, ranking it 179th of 189 countries by the Human Development Index [20]. Over 40% (33 million) of its people have no access to quality water, despite the fact that over 50% of Africa’s water reserves are located in the DRC. Less than 10% of the population has access to electricity. Over 70% of its people lack adequate food, including children under age 5, 50% of whom are affected by chronic malnutrition and 1.3 million by severe acute malnutrition [21]. Only 35% of children aged 12–23 months are fully vaccinated before their first birthday, with 20% receiving no vaccinations at all. The country suffers from a severe shortage and inequitable distribution of healthcare personnel. There are 0.28 physicians and 1.91 nurses and midwives per 10,000 population, with the ecancer 2021, 15:1231; www.ecancer.org; DOI: https://doi.org/10.3332/ecancer.2021.1231 2 Research Introduction Against this backdrop of economic deprivation, political instability and infectious disease epidemics, a cancer crisis is also unfolding in the DRC. Cancer is among the leading causes of death worldwide. In 2018, there were 18.1 million new cancer cases and 9.5 million cancerrelated deaths globally, far exceeding the number of deaths from human immunodeficiency virus/AIDS, malaria and tuberculosis combined [27]. World Health Organization (WHO) predicts that by 2040, the annual number of new cancer cases in the world will rise to 29.5 million and cancer-related deaths to 16.4 million [28]. While an estimated 60% of cancer cases and 75% of deaths will occur in low- and middleincome countries [29], only 5% of global spending [30] on cancer is directed to these countries. Two cancers of particular importance are those originating in the cervix and breast, as their incidence rates are rising, mortality rates are among the highest in the world and together constitute half of all new cases of cancer in women in the African region [31, 32]. It is predicted that without interventions, the annual number of new cases and related deaths from these two cancers will more than double by 2040 [32]. Among all new adult malignancies diagnosed in the DRC in 2018, cancers of the breast (12.6%) and cervix (11.8%) were the most common, together accounting for almost half of the 27,000 cancers diagnosed in females [33]. This is almost certainly a major underestimation as case ascertainment is poor in urban settings and non-existent in most of the rural areas of the country. Although cervical cancer is the number one cause of cancer-related death among Congolese adults [33], public awareness about the disease remains extremely low. In a recent cross-sectional study of 524 women aged 16–78 years (median age: 28 years), in the capital city of Kinshasa, <20% of women interviewed knew the cause of cervical cancer or how it could be prevented or treated; <10% had ever had a Pap (cytology) smear and >70% used plants or chemicals for vaginal care [34]. With the common goal of improving healthcare in Africa, a partnership was formed between the Dikembe Mutombo Foundation (DMF) [35] and the Friends of Africa, Inc. (FOA) [36], both US 501(c)3 organisations. Founded and led by the US National Basketball Association legend Dikembe Mutombo, originally from the DRC, the DMF is dedicated to improving the health, education and quality of life for the people of the DRC. The organisation strives to accomplish its mission by promoting primary health care and disease prevention, health policy, research and increased access to health care education. In 2007, to help realise its goals, the DMF constructed a privately-owned, 150-bed primary care hospital – Biamba Marie Mutombo Hospital (BMMH) – in central Kinshasa. The FOA is an organisation primarily composed of US trained and board-certified surgical oncologists committed to reducing cervical and breast cancer mortality in Africa and its Diaspora. Founded in 1989, the FOA (1) develops training programmes and clinical services for the early detection of women’s cancers; (2) sponsors onsite, handson surgical oncology education seminars; (3) promotes local ownership of medical clinics and supply chains and (4) supports practical and relevant scientific research activities. Our primary objective was to increase access to women’s cancer (cervix and breast) services in the DRC by building a contextually-appropriate and sustainable diagnostic and therapeutic service platform. Methods Situational assessment Consensus meeting The first step in assessing the local conditions and circumstances was to convene a broad cross section of stakeholders for a Consensus Meeting. In March 2016, key Congolese stakeholders from the government, civil society, private and public health sectors and academia were convened over a 3-day period in the capital city of Kinshasa. The format of the meeting consisted of formal presentations of peer-reviewed, gray literature and unpublished data on the following topics: (1) epidemiology of women’s cancers in the African region with a focus on the DRC, [33, 34, 37–45] and (2) current cancer care workforce capacity, clinical services and infrastructure in the DRC [46, 47] (Table 1). ecancer 2021, 15:1231; www.ecancer.org; DOI: https://doi.org/10.3332/ecancer.2021.1231 3 Research northern rural areas of the country having the fewest health workers, particularly physicians (1.1%–2.6%) [22]. Few students from any of the 39 nationally recognised medical schools have the opportunity to spend the required years in clinical rotations due to a lack of participating hospitals. The overall result is an education of doubtful quality [23, 24]. Residency training is rare with approximately 2.5% of the rural-school graduates and 15.2% of urban-school graduates entering formal residency programmes. Deficiencies in the public health system are central to the recent outbreaks of measles, circulating vaccine-derived polioviruses type 2 and cholera in 2019, and Ebola a year earlier [25, 26]. Hospital site visit Following the Consensus Meeting, a site visit was conducted at BMMH by a visiting US gynaecologic oncologist (Groesbeck Parham). Discussions were held with hospital administrators, clinicians (gynaecologists, surgeons, nurses, anaesthesiologists), clinical managers, procurement specialists, data managers and DMF leadership. An assessment was made of the physical plant, clinical capacity, infrastructure and interest among staff in participating in a women’s cancer care initiative. Implementation strategy Informed by the findings from the situational assessment, a team of US and Zambian surgical and clinical oncologists and nurse specialists was tasked by the FOA to design an implementation strategy that encompassed training a local oncology workforce, developing clinical services and building a clinical infrastructure for women’s cancer care services. Results Stakeholders attending the Consensus Meeting listed the following as major barriers to the implementation of women’s cancer care services in the DRC: (1) low levels of cancer awareness among health professionals and the general population; (2) lack of trained oncology human resources of all types, including ancillary personnel; (3) lack of financing and unreliable systems for the procurement, storage and dispersal of equipment and supplies; (4) poor access to affordable cancer medicines and (5) properly outfitted clinics and surgical theatres (Table 1). Implementation strategy Workforce capacity building The above findings, coupled with a wide variation in the backgrounds and clinical experiences of the designated BMMH staff-trainees, prompted the FOA team to adopt competency-based training enfolded into a matrix of self-directed learning and bedside mentoring as the strategy for workforce capacity building. Mastery of the various clinical and surgical procedures was facilitated by using the following skillsbuilding techniques: deconstruction (each procedure was broken down into its subcomponents); high volume repetition (repeating the same clinical procedures over short time intervals); pre- and post-case review and mental narration (imaginary enactment of clinical procedures). South-south professional exchange programmes were also felt to represent potential opportunities for skill building. Onsite mentoring was led by the visiting FOA oncology team on an intermittent basis, as conditions permitted. Clinical services Given the low socioeconomic status of the target population and their lack of access to cancer prevention and treatment services, the clinical services deemed to be of greatest value were the following: (1) cervical cancer screening using visual inspection with acetic acid (VIA) and digital cervicography; (2) treatment of cervical cancer precursors using thermal ablation and large loop excision of the transformation zone (LLETZ); (3) evaluation of symptomatic women with clinical breast exam, breast ultrasound and ultrasound-guided core needle biopsy/fine needle aspiration; (4) surgical treatment of cervical and breast cancers; (5) breast oncoplastic procedures; (6) palliative surgery and (7) infusion of chemotherapy for advanced cervical and breast cancers (Table 1). Infrastructure investments Investments were made in outpatient clinics and surgical theatres to ensure that the trainers and trainees had well-equipped clinical facilities for both teaching and clinical care, respectively. Dedicated space for the new clinical services was assigned by the hospital administration, after which it was remodelled and furnished with the necessary equipment, supplies and furniture (Table 2). ecancer 2021, 15:1231; www.ecancer.org; DOI: https://doi.org/10.3332/ecancer.2021.1231 4 Research Situational assessment Table 1. Assessment and training. Outcomes Rapid situational assessment of the status of women’s cancer services in the DRC • Consensus Meeting of key stakeholders from government, civil society, business and academia • Onsite assessment of activities and human resources of BMMH • Gaps, needs and solutions identified Awareness activities – Cervical/breast cancer • Cervical and breast cancer public awareness campaigns in DRC (Kinshasa) • Television and radio announcements • Community-based awareness events Training activities – Cervical cancer • Hands-on training for cervical cancer prevention in Zambia and DRC • BMMH staff doctors and nurses trained to perform ‘single-visit cervical cancer screening and treatment’ by visiting Zambian nurse experts • On-site surgical training in DRC • BMMH staff gynaecologists trained to perform radical abdominal hysterectomy and bilateral pelvic lymphadenectomy by visiting US gynaecologic oncologists Training activities – Breast cancer • On-site clinical and surgical training in DRC • BMMH staff general surgeons and nurses trained to evaluate breast masses and axilla with ultrasound and ultrasound-guided core-needle biopsy/fine needle aspiration by visiting US breast oncologist • BMMH staff general surgeons trained to perform lumpectomy and modified radical mastectomy and axillary LNDa, palliative and breast oncoplastic procedures by visiting US breast oncologist Training activities – Chemotherapy • Hands-on short course in chemotherapy administration in Zambia and DRC • BMMH staff physicians trained in principles and practice of safe administration of chemotherapy for cervical and breast cancer by clinical oncologists at Zambia’s national cancer centre Research Activities *aLND, Lymph node dissection Timeline The initial plan was to implement workforce training, clinical service and infrastructure development through quarterly visits over a 12-month period. The intended timeline was significantly altered from 12 to 24 months (January 2017–December 2019) due to travel restrictions related to multiple episodes of political unrest within the DRC and Ebola outbreaks. Mentorship was continued in between visits through electronic communication [9]. Clinical outcomes Cervical cancer Following approval by the DRC Ministry of Health, the DMF and FOA, in collaboration with the United Nations Population Fund, co-sponsored a cervical cancer awareness campaign in Kinshasa (May–June 2016). Awareness activities, led by teams of nurses and doctors from the ecancer 2021, 15:1231; www.ecancer.org; DOI: https://doi.org/10.3332/ecancer.2021.1231 5 As of January 2020, 28,417 women have been screened for cervical cancer, of which 2,153 (1 of every 13 screened) were found to have cervical abnormalities suggestive of cervical pre-cancer. More than 90% were treated on the same day with either thermal ablation or LLETZ. Invasive cervical cancer was pathologically confirmed in 284 (1%) of the screened population (Figure 1). of which 48 (17%) patients were early stage and operable. Patients with advanced stage invasive cancers were referred to the national cancer centre in Zambia for radiation therapy. Those who could not afford radiation therapy in Zambia were put on a palliative care regimen, as there were no chemotherapy or radiation therapy facilities in the DRC during the time of programme implementation (Figure 1). Table 2. Infrastructure investments. Types • Details Health services • Cervical cancer prevention clinic • Single-visit screening and treatment services • Cervical cancer surgical unit • Surgical management of early-stage cervical cancer • Breast cancer clinic • Clinical breast examination, diagnostic ultrasound, core-needle biopsy • Breast cancer surgical unit • Surgical management of breast cancer, including oncoplastic procedures • Chemotherapy suite • Administration of chemotherapeutic agents to treat breast and cervical cancer Human resources • Cervical cancer ‘screen and treat’ providers (5) • Gynaecologists trained to perform radical abdominal hysterectomy and lymphadenectomy (3) • General surgeons trained to perform breast conservation and palliative surgery, and oncoplastic procedures (2) • General surgeons (2) general physicians (2) and nurses (3) trained to perform breast diagnostics • General physicians (2) trained to administer chemotherapy • Procurement officer (1) • Data manager (1) Equipment and supplies • Procured clinic and operating room equipment and supplies (ultrasound machines, examination tables, core needle biopsy, surgical instruments, suture material, gynae examination equipment, thermal ablation and loop electrosurgical excision procedure /LLETZ, digital cameras, computers, smart phones, storage facilities for chemotherapy drugs, personal protective equipment, chemotherapy hood) ecancer 2021, 15:1231; www.ecancer.org; DOI: https://doi.org/10.3332/ecancer.2021.1231 6 Research BMMH, were comprised of television and radio announcements and talk shows, combined with community-based educational sessions at large public gathering sites (markets and religious services) throughout the city. Immediately following the campaign, a small team of BMMH staff doctors and nurses attended a hands-on cervical cancer prevention educational seminar at the University Teaching Hospital in Lusaka, Zambia (20 June–1 July 2016), where they were trained to perform ‘single-visit cervical cancer screening and treatment’ procedures using VIA, digital cervicography, thermal ablation and LLETZ [13]. Upon completion of their training, they returned to the DRC and established the BMMH Cervical Cancer Early Detection and Treatment Clinic, based on the Zambian model [13]. A cervical cancer ‘screen and treat’ camp was announced on radio and television then held at the BMMH (13—18 July 2016). During the week-long event, over 8,000 women presented for cervical cancer screening services. Screening was conducted by the newly trained BMMH team during the week-long camp, under the direct supervision of cervical cancer prevention nurse-trainers from Zambia. Clients found to have precancerous cervical lesions were offered same-day treatment using either thermal ablation or LLETZ by the BMMH staff. Those whose cervical lesions were grossly suspicious for invasive cancer were evaluated with punch biopsy. If pathologically confirmed to have early-stage invasive cancer, they were counselled and offered surgery by the visiting team of US surgical oncologists (gynaecologic oncologists), during which time BMMH staff gynaecologists were taught how to perform radical abdominal hysterectomy, bilateral pelvic lymphadenectomy [9]. All women who underwent screening were also asked by screening nurses if they had breast symptoms. Those who did were referred to the newly opened BMMH Breast Clinic for further evaluation. Research Figure 1. Cervical cancer programme outcomes. Breast cancer Following the initial cervical cancer screening camp in July 2016, four additional visits were made to the DRC by the FOA team of mentors from January 2017–December 2019. During these visits, they were joined by a US breast surgical oncologist (Ronda Henry-Tillman) who implemented a hands-on training curriculum for BMMH staff general surgeons and nurses, designed to teach clinical breast examination, ultrasound imaging of the breast and axilla ultrasound-guided core needle biopsies and fine need aspirations. A surgical training curriculum for staff general surgeons was initiated by the US breast surgical oncologist with the goal of building their capacity to perform breast lumpectomy, modified radical mastectomy, axillary node dissections, breast oncoplastic procedures and palliative surgery. The curriculum for teaching breast diagnostic and surgical procedures was previously developed and implemented in Zambia [12]. As of December 2019, 5,211 women have been evaluated using clinical breast examination and breast ultrasound. The vast majority presented with symptoms, elicited by nurses during cervical cancer screening. Clinical abnormalities were detected in 1,420 (27%) women, of which 516 (36%) met the criteria for ultrasound-guided core needle biopsy. Pathology reports were available on 368 (71%), of which 164 (44%) were malignant and 204 (55%) benign. Approximately 90% of the breast cancers were advanced stage (stage 3 or 4) (Figure 2). A total of 241 women have undergone surgery, consisting of 183 breast lumpectomies, 58 modified radical mastectomies and 48 axillary lymph node dissections (LND) (Figure 2). The specifics of this programme and outcomes are described in the accompanying breast manuscript [17]. Chemotherapy Some of the chemotherapeutic agents indicated for the treatment of breast cancer were available for purchase from local private vendors in Kinshasa; however, their prices made them unaffordable for the vast majority of patients. Neither was there a mechanism for verifying their quality. Access to cytotoxic cancer medicines at a discounted price became available through a partnership with the Bioventures for ecancer 2021, 15:1231; www.ecancer.org; DOI: https://doi.org/10.3332/ecancer.2021.1231 7 Global Health (BVGH) African Access Initiative [48], a nonprofit organisation that expands access to cancer medicines and technologies in Africa through a sustainable access model. To ensure the quality of the cancer drugs, only companies whose products are United States Food and Drug Administration-approved are invited to participate. The partnership with BVGH created opportunities for the development of a chemotherapy unit at BMMH. To satisfy the need for training, an intense, hands-on, short-course (6 weeks) in chemotherapy administration was developed at Zambia’s national cancer centre – Cancer Diseases Hospital – tailored for BMMH health professionals. Under this curriculum two BMMH general physicians and three nurses were trained to safely and effectively administer chemotherapy for breast and cervical cancer, following strict protocols. Upon course completion, the trainees returned to the DRC and established the BMMH Chemotherapy Infusion Unit in June 2019. Guided by clinical oncologists from Zambia’s national cancer centre, the infrastructure for the chemotherapy was established. Oversight and quality assurance were provided through a combination of onsite visits and teleconferencing with a senior chemotherapy nurse and clinical oncologist from the Zambian cancer centre. This experience is described in detail in the chemotherapy manuscript in this series [16]. Since forming a partnership with BVGH, over 100 breast and cervical patients have been safely treated with chemotherapy, having received a total of 377 cycles of cytotoxic agents. This model of purchasing chemotherapy drugs at a reduced cost is one of the only options for accessing essential cancer medicines, irrespective of whether they exist on national formularies. For more details, refer to the chemotherapy manuscript in this series [16]. Human resources Discussion We report one of the few real-world efforts to implement detection and treatment services for cervical and breast cancer in a fragile nation torn by war, outbreaks of infectious diseases, political unrest and devastating poverty. Figure 2. Breast cancer programme outcomes. ecancer 2021, 15:1231; www.ecancer.org; DOI: https://doi.org/10.3332/ecancer.2021.1231 8 Research A wide variety of 21 BMMH staff members were trained during the course of the initiative (Table 2). Using a unique matrix of self-directed learning, onsite and regional hands-on training seminars, south-south professional exchange programmes and teleconferencing, Congolese-led women’s cancer services were established in a private healthcare facility in Kinshasa. Skills transfer was facilitated through a process of clinical and surgical bedside mentoring, rendered by a close-knit team of highly skilled universitybased US and Zambian oncologists and Zambian nurse-specialists who had a history of working together in resource-constrained African environments. Through wise investments, the physical and clinical infrastructure needed to support the newly trained workforce was simultaneously established. In between onsite visits, modern forms of telecommunication (e.g., Zoom teleconferencing) were used to facilitate continuing medical education. Central to the success of the effort was the support of the hospital’s administrative staff and the sponsoring organisation (DMF), coupled with the leadership, clinical knowledge and logistical guidance provided by local professional colleagues. Meaning and context Building the pathway for cancer care in severely resource-constrained settings is a difficult task, as it involves the integration of a cancer care platforms into healthcare systems whose supportive services (blood banking, laboratory, procurement, pathology, pharmacy, etc.) are often quite meagre. Yet, these are the circumstances under which one must operate when faced with the task of implementing cancer care services in the world’s poorest nations. When working in settings that are severely resource-constrained, a key ethical question that must be addressed is whether attempts should be made to implement only certain parts of the cancer care pathway if the system cannot be implemented in its entirety. In our particular situation, the outstanding question was whether cancer detection, diagnostic and surgical services should be implemented in the absence of radiation therapy services, given the likelihood of uncovering large numbers of cases of advanced disease that would require these adjunctive therapeutic modalities. When posed to key local stakeholders and organisations in attendance at the Consensus Meeting, the consensus opinion was to begin, immediately, by training the workforce and building the clinical services and infrastructure needed for delivery of early detection and surgical treatment services. The underlying rationale was that the present inability to treat all cancer cases should not impede efforts to prevent those cases that can be prevented (e.g. ablation of precancerous conditions of the cervix), treat those cases that can be treated (e.g. early-stage cancers that can be cured with surgery alone) and relieve suffering in the others. These recommendations were in line with the humanitarian principles embedded in the ‘Four Pillars of Oncology’: prevent all cancers that can be prevented; treat all cancers that can be treated; cure all cancers that can be cured; provide palliation whenever palliation is required [53]. There was also general consensus that efforts should be pursued to fill the remaining gaps in the clinical care pathway by addressing the barriers and establishing access. Motivated by a commitment to actively engage the problem by implementing contextually-appropriate and evidence-based solutions, and to learn on the go, we started with the resources that were available. We made frequent use of the various sets of resource-stratified management guidelines for cervical and breast cancer developed by the WHO, Breast Health Global Initiative, National Comprehensive Cancer Network and American Society of Clinical Oncology [54–57]. Similar sets of rigorously examined strategies for implementation in resource-constrained settings are lacking, particularly as it pertains to FCV settings. In developing an implementation strategy, we relied on our understanding of the natural history of the diseases, accumulated experience working as clinicians in low-income African countries and underserved communities in high-income countries and a clear-headed assessment of what could be done safely, effectively and in a sustainable fashion. In-country and regional (south-south) trainings were emphasised, as opposed to training in high-income countries, which can lead to translation difficulties because of vast contextual differences. The decision to execute training and diagnostic and treatment services within a private versus public sector facility served to circumvent some of the logistical problems we might have confronted in the public sector, such as lack of personal security, bureaucratic inefficiencies, unreliable procurement systems and the potential for politicisation and militarisation of health services. ecancer 2021, 15:1231; www.ecancer.org; DOI: https://doi.org/10.3332/ecancer.2021.1231 9 Research Fragility and violent conflict are on the rise worldwide. When protracted, their consequences extend beyond the war-wounded, impacting the health of entire communities. By 2030, up to two-thirds of the global extreme poor will live in societies affected by fragility, conflict and violence [1]. Noncommunicable diseases, including cancer, are increasing in these settings [49, 50], and women are disproportionately impacted. Of the 2 million annual new cases of cervical and breast cancer and 800,000 annual cancer-related deaths that occur in the world, a significant percentage are in women who live in the fragile and conflict ecosystems of sub-Saharan Africa [1], where opportunities for cancer care are limited [51, 52]. Limitations Cancer care as a human right is in accordance with human rights instruments adopted by the United Nations Human Rights Council [58]. We recognise the tension between a private sector model that rests on a fee-for-service platform in a setting in which there are no governmentsupported social insurance schemes. However, even when services appear to be ‘free’, they are in fact subsidised or paid for by external or even internal philanthropy, e.g., substantial fees paid by private patients to cover the expenses of those who are unable to pay. The ultimate answer is a well-financed Universal Health Care insurance scheme funded by local governments, as outlined in the United Nations 2030 Agenda for Sustainable Development [59, 60]. By attaching cancer control services to a fee-for-service healthcare facility, fewer people will be able to afford them but the chances they will be sustained may be increased, particularly in fragile and conflict-affected settings. Challenges and solutions Some of the challenges encountered and solutions derived during the course of our work were as follows: In order to increase the availability and accessibility of cancer detection services, quarterly week-long mobile outreach cervical cancer early detection camps were sponsored by the BMMH. To date, six such outreach camps have been held, all in densely populated areas around Kinshasa, led by hospital nursing staff, through which over 5,000 women have been screened for cervical cancer. Three of the outreach camps were mentored, onsite, by visiting Zambian nurse-trainers. High cost of pathology services The costs of pathology services were unaffordable for most patients, resulting in significant delays, and impacting timely treatment. As a short-term solution, we negotiated reduced fees with private sector pathologists in Kinshasa, however, most patients still had difficulty paying the residual costs. These circumstances helped to motivate the formation of a partnership between the DMF and American Society of Clinical Pathology, resulting in a venture to build chemistry and pathology laboratory infrastructure at the BMMH. All equipment has been purchased, installed and a local pathologist hired to lead the programme. Expectations are that the laboratory will be fully operational in April 2021. Access to cancer medicines The World Health Organization has an Essential Medicines List that contains several chemotherapeutic and palliative agents used in the treatment and palliation of breast and cervical cancer [61]. Other than tamoxifen, these medicines are difficult to acquire or are unaffordable for governments or patients in many sub-Saharan African countries. Purchasing chemotherapy, even at low cost, inevitably leads to resource limitations in that it is not sustainable. However, if properly managed, it can be creatively leveraged to generate profit that can be used to support future purchases. We were able to partner with an international non-profit global health organisation (BVGH) that facilitates access to selected cancer medicines at a reduced cost [48]. Lack of radiation therapy services Radiation therapy is an essential pillar in the treatment of cervical and breast cancer. Early-stage cervical cancers with low disease volume can often be cured with surgery alone. Others require adjunctive treatment with radiation. Radiation therapy is also essential in the management of breast cancer patients who are treated with conservation therapy as well as those with advanced disease. We were aware that we would encounter patients that could not be optimally treated because of the lack of radiation therapy. This problem is not just limited to the DRC ecancer 2021, 15:1231; www.ecancer.org; DOI: https://doi.org/10.3332/ecancer.2021.1231 10 Research Supply/demand imbalances for screening services but across the region as only 23 of 52 African countries are known to have radiation therapy facilities [62]. This was the central dilemma in our decision to start with what was available instead of waiting until all components in the cancer care pathway were in place. However, not to begin with the resources we had at hand would lead to continued missed opportunities to prevent cancers and cure the patients that we could, and maybe prolong the lives of others. Towards that end, present discussions are underway to build a collaborative relationship between BMMH and a new privately-owned radiation therapy centre in Kinshasa. Implications for the future Private enterprises can help break the cycle of fragility, conflict and poverty. Businesses create jobs and income and support livelihoods, and in doing so can contribute to social cohesion and stability. Private investments in cancer care in resource-constrained environments deserve further consideration [63]. This marriage may have the best chance of success in a private, non-profit institution like the BMMH where the ultimate goal is to balance the economic capacity of the community with the financial resources required to deliver a high quality of care and ensure institutional sustainability. Conclusion Using a novel partnership consisting of oncology health professionals from Africa and its Diaspora, international philanthropic organisations, a cancer medicine access initiative and an established African cancer centre, we successfully trained the workforce and built the clinical services and infrastructure for women’s cancer care services in a private sector health facility in the DRC. List of abbreviations BMMH, Biamba Marie Mutombo Hospital; BVGH, Bioventures for Global Health; DMF, Dikembe Mutombo Foundation; DRC, Democratic Republic of the Congo; FCV, Fragile, Conflict and Violence; FOA, Friends of Africa, Inc.; HPV, Human papilloma virus; LLETZ, Large loop excision of the transformation zone; LND, Lymph node dissection; VIA, Visual inspection with acetic acid; WHO, World Health Organization. Conflicts of interest None of the authors declare any conflicts of interest. ecancer 2021, 15:1231; www.ecancer.org; DOI: https://doi.org/10.3332/ecancer.2021.1231 11 Research Limited, one-time investments such as ours can be very beneficial in that they can provide the capital outlay for training and infrastructure development that can be continually realised. The presence of women’s cancer care at BMMH can now be used to offset expenses by offering services at affordable but profitable fees. It can also be presented as a model of cancer care to investors interested in promoting the growth of for-profit health enterprises in low-income nations, particularly those affected by conflict and violence. Services can be leveraged to advocate for government-led national health promotion and awareness campaigns for cervical and breast cancer control; public sector breast and cervical cancer early detection and treatment services; national Human Papilloma Virus vaccination programmes for adolescent girls; expansion of national laboratory and pathology services; public sector radiation therapy services; partnerships with pharmaceutical firms to help ensure access to essential cancer medicines and the development of a national strategic plan for cancer control. Acknowledgments The authors acknowledge: • Dikembe Mutombo Foundation Friends of Africa, Inc. • Nurses and oncologists: University of Zambia and Cancer Diseases Hospital, Zambia • Administration and clinical staff: Biamba Marie Mutombo Hospital • BIO Ventures Global Health Funding statement Funding for the initiative was provided by a generous grant from the Howard G. 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Original Research 06 March 2023 10.3389/fphys.2023.1090937 TYPE PUBLISHED DOI OPEN ACCESS EDITED BY Jieyun Bai, Jinan University, China REVIEWED BY Pranjali Gajbhiye, Nirvesh Enterprises Private Limited, India Louise Ghesquière, Centre Hospitalier Regional et Universitaire de Lille, France *CORRESPONDENCE Zhidong Zhao, zhaozd@hdu.edu.cn A lightweight fetal distress-assisted diagnosis model based on a cross-channel interactive attention mechanism Yanjun Deng 1, Yefei Zhang 1, Zhixin Zhou 1, Xianfei Zhang 1, Pengfei Jiao 2 and Zhidong Zhao 2* 1 School of Electronics and Information, Hangzhou Dianzi University, Hangzhou, China, 2School of Cyberspace Security, Hangzhou Dianzi University, Hangzhou, China SPECIALTY SECTION This article was submitted to Computational Physiology and Medicine, a section of the journal Frontiers in Physiology 06 November 2022 10 February 2023 PUBLISHED 06 March 2023 RECEIVED ACCEPTED CITATION Deng Y, Zhang Y, Zhou Z, Zhang X, Jiao P and Zhao Z (2023), A lightweight fetal distress-assisted diagnosis model based on a cross-channel interactive attention mechanism. Front. Physiol. 14:1090937. doi: 10.3389/fphys.2023.1090937 COPYRIGHT © 2023 Deng, Zhang, Zhou, Zhang, Jiao and Zhao. 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. Fetal distress is a symptom of fetal intrauterine hypoxia, which is seriously harmful to both the fetus and the pregnant woman. The current primary clinical tool for the assessment of fetal distress is Cardiotocography (CTG). Due to subjective variability, physicians often interpret CTG results inconsistently, hence the need to develop an auxiliary diagnostic system for fetal distress. Although the deep learning-based fetal distress-assisted diagnosis model has a high classification accuracy, the model not only has a large number of parameters but also requires a large number of computational resources, which is difficult to deploy to practical end-use scenarios. Therefore, this paper proposes a lightweight fetal distressassisted diagnosis network, LW-FHRNet, based on a cross-channel interactive attention mechanism. The wavelet packet decomposition technique is used to convert the one-dimensional fetal heart rate (FHR) signal into a two-dimensional wavelet packet coefficient matrix map as the network input layer to fully obtain the feature information of the FHR signal. With ShuffleNet-v2 as the core, a local cross-channel interactive attention mechanism is introduced to enhance the model’s ability to extract features and achieve effective fusion of multichannel features without dimensionality reduction. In this paper, the publicly available database CTU-UHB is used for the network performance evaluation. LW-FHRNet achieves 95.24% accuracy, which meets or exceeds the classification results of deep learning-based models. Additionally, the number of model parameters is reduced many times compared with the deep learning model, and the size of the model parameters is only 0.33 M. The results show that the lightweight model proposed in this paper can effectively aid in fetal distress diagnosis. KEYWORDS fetal distress, fetal heart rate, lightweight model, attention mechanism, wavelet packet coefficient 1 Introduction Fetal distress is a syndrome of respiratory and circulatory insufficiency caused by intrauterine fetal hypoxia during labor and is closely associated with changes in fetal heart rate signals (Blickstein and Green, 2007; Spairani et al., 2022). Fetal distress may cause hypoxic-ischemic encephalopathy and eventually leading to cerebral palsy or perinatal death (Bobrow and Soothill, 1999). Early detection and diagnosis of fetal distress can help prevent damage to the vital organs of the fetus prior to delivery. Therefore, it is important to enhance Frontiers in Physiology 01 frontiersin.org Deng et al. 10.3389/fphys.2023.1090937 beginning to make their mark in the medical signaling field. Cao et al. (2021) proposed a multichannel lightweight model with each channel integrating multiple heterogeneous convolutional layers to obtain multilevel features for classifying myocardial infarction with an accuracy rate of 96.65%. Zheng et al. (2021) trained MobileNetV1 and MobileNetV2 models by migration learning for pterygium diagnosis in the eye and compared them with the classical model and found that MobileNetV2 obtained better results with a model size of only 13.5 M. Chen et al. (2022) used the lightweight networks MobileNetV1, MobileNetV2, and Xception to classify cervical cancer cells and used knowledge distillation for accuracy improvement. Among them, Xception matched the accuracy of the large network Inception-ResNetV2, while the model size was only 40%. The lightweight network model effectively reduces the number of model parameters and opens up a method for promoting a low-cost operating model. However, the feature extraction ability and the network classification accuracy still need to be further improved. Aiming at the complexity and considerable computation in existing deep learning-based fetal distress algorithm models, this paper introduces a lightweight network architecture to design a lightweight fetal distress-assisted diagnosis network based on FHR. Additionally, to further improve the feature extraction ability and classification effect of the network, the attention mechanism is incorporated into the lightweight network to build a lightweight network unit (ECA-Shuffle) based on the cross-channel interactive attention mechanism. The main contributions of this paper are as follows. intrauterine fetal status monitoring during pregnancy to ensure the safety of the fetus and the pregnant woman. The most common method for monitoring fetal status in clinical practice is CTG monitoring (Grivell et al., 2015). The CTG signal consists of the FHR curve and uterine contraction (UC) curve. Through CTG monitoring, doctors can detect fetal distress in time so that they can take effective treatment measures to protect the health of the fetus. However, the diagnosis is too dependent on physician experience and interobserver disagreement when interpreted by the physician’s naked eye alone (Bernardes et al., 1997; Palomaki et al., 2006). Therefore, there is an increased incidence of unnecessary cesarean section due to subjective physician error (Abdulhay et al., 2014; Marques et al., 2019). With the development of artificial intelligence technology, scholars worldwide are committed to developing fetal healthassisted diagnosis systems based on machine learning and deep learning to help healthcare professionals analyze CTG signals objectively and correctly. Barquero-Perez et al. (2017); Spilka et al. (2014); Georgoulas et al. (2017); Yilmaz. (2016) used normalized compression distance, random forest (RF), support vector machine (SVM), and artificial neural network (ANN) classification algorithms, respectively, to classify CTG signals for fetal distress problems and achieved good results. Zhao et al. (2018) extracted 47 features from different domains (morphological, time domain, frequency domain and non-linear domain) and selected Decision Tree, SVM and adaptive boosting, respectively, for fetal acidosis classification. Comert et al. (2018) used short-time Fourier transform (STFT) to obtain 2-D images and combined it with transfer learning and convolutional neural networks to predict fetal distress (Liu et al., 2021). proposed an attention-based CNN-BiLSTM hybrid neural network enhanced with features of discrete wavelet transformation, obtaining an average sensitivity, specificity and quality index of 75.23%, 70.82%, and 72.93%, respectively. Zhao et al. (2019) used recurrence plot to convert one-dimensional FHR to two-dimensional and fed into convolutional neural network to obtain 98.69% accuracy in fetal distress classification. Baghel et al. (2022) obtained 99.09% classification accuracy by performing direct 1-D convolutional operations on the FHR signal after Butterworth filtering. Although the abovementioned classification models based on machine learning and deep learning achieve better results, the complexity of the model and the large number of parameters take up large computational resources, which leads to the model being highly dependent on the performance of the device hardware and difficult to deploy to the terminal for generalized application. Lightweight models and miniaturization have become a trend in many application scenarios, so an increasing number of academics are focusing on lightweight network models that can be deployed and run directly on mobile devices. The MobileNet series (Howard et al., 2017; Sandler et al., 2018; Howard et al., 2019) and ShuffleNet series (Ma et al., 2018; Zhang et al., 2018) of lightweight networks currently have good performance in the target detection and image classification field. MobileNet model is a lightweight deep neural network proposed by Google for embedded devices, using the core idea of depthwise separable convolution. ShuffleNet model is a neural network structure designed for devices with limited computational resources, mainly using pointwise group convolution and channel shuffle. Lightweight models are also Frontiers in Physiology (1) The matrix feature map based on wavelet packet coefficients is constructed to refine the FHR signal in multiple frequency bands and used as input to the model. Different wavelet basis functions are selected to generate multiple feature maps to vote on the sample classification results. (2) The cross-channel interactive attention module is embedded in the tail of the ShuffleNet-V2 base unit to generate an ECAShuffle unit to achieve effective multichannel feature fusion without dimensionality reduction. (3) A lightweight fetal distress-assisted diagnosis network based on the FHR signal, LW-FHRNet, is proposed. Conventional convolution with ECA-Shuffle units ensures effective channel feature fusion while reducing model complexity and enhances the model’s ability to classify fetal distress. The rest of the paper is presented below. Section 2 describes the overall scheme in detail. Section 3 describes the database, experimental setup and results in detail. Section 4 discusses and analyzes the performance of the proposed model. The final section contains conclusions and future work. 2 Materials and methods The architecture of the lightweight fetal distress-assisted diagnosis model based on the cross-channel interactive attention mechanism designed in this paper is shown in Figure 1, including a preprocessing module, a feature map construction module, and a feature extraction and classification module. First, the missing values 02 frontiersin.org Deng et al. 10.3389/fphys.2023.1090937 FIGURE 1 Description of the architecture for the proposed lightweight network-based fetal distress assisted-diagnosis model. (3) If the FHR value is greater than 200 bpm or less than 50 bpm, it is filled in with Hermite spline interpolation. and spikes in the FHR signal are removed by signal preprocessing, and the signal is segmented into 20-min lengths. Second, the wavelet packet decomposition technique is used to construct wavelet coefficient matrix feature maps of FHR signals based on db1 to db5 wavelet basis functions. Finally, LW-FHRNet is constructed by using deep separable convolution, channel shuffle and other techniques and incorporating a local cross-channel interactive attention mechanism without dimensionality reduction, which effectively reduces the number of model parameters and improves the classification accuracy of the model. Noise and missing value segments in the FHR signal can be effectively filtered out by the above interpolation method. In conjunction with the time requirement of clinical prenatal examination, this paper uses 20-min data segments for analysis. The preprocessed data are segmented into 20-min time segments to obtain multicomponent segment data. The waveform obtained using the above preprocessing method is shown in Figure 2, where (a) is the raw data of the FHR signal, (b) is the waveform after preprocessing using the above method, and (c) is the segment after splitting the data into multiple segments with a 20-min data length. 2.1 Signal preprocessing Clinically, the FHR signal is acquired mainly by an ultrasound Doppler probe placed in the abdomen of the pregnant woman. During the acquisition process, the signal is inevitably subject to a variety of noise interferences, such as the movement of the fetus and the pregnant woman, improper placement of the sensor and other external factors. The noise of the FHR is represented by spikes (FHR values greater than 200 or less than 50 bpm) and missing values (FHR values equal to 0) (Cesarelli et al., 2007). Accordingly, the purpose of preprocessing is to remove these two types of noise. In this study, the interpolation method is used to remove noise (Chudaek et al., 2009), and the specific process is as follows. 2.2 Construction of feature maps based on wavelet packet coefficients As a non-stationary and non-linear time series, FHR contains complex physiological and pathological information. Wavelet packet decomposition is a discrete analysis method of non-stationary signals that can select the appropriate spectral band according to the signal characteristics and improve the time-frequency analysis resolution (Behera and Jahan, 2012). In this paper, wavelet packet decomposition is introduced to construct the wavelet packet coefficient matrix using different subspace coefficients to convert the 1D FHR signal into a 2D wavelet packet coefficient feature map. The feature map is used as the input layer data for the deep network model. Figure 3A shows the wavelet packet coefficient matrix construction process. The signal is decomposed into (1) If the FHR value is equal to 0 and the duration is greater than 15 s, the segment is removed directly; otherwise, it is linearly interpolated. (2) If the FHR value is unstable, i.e., the absolute value of two adjacent points is greater than 25 bpm, and interpolation is performed between the starting sampling point and the first point of the next stabilization segment. A stable segment is defined as five consecutive FHR values where the difference is less than 10 bpm. Frontiers in Physiology 03 frontiersin.org Deng et al. 10.3389/fphys.2023.1090937 FIGURE 2 FHR signal preprocessing process. Remove spikes and missing values of the original signal, then divide into segments of 20-min length. (A) The original signal, (B) processed signal, (C) segmented signal. FIGURE 3 Construction of feature maps based on wavelet packet coefficient matrix. (A) Construction of wavelet packet coefficient matrix; (B) Construction of db1~db5 feature map. 1 h(k)  √
〈φ(t), φ(2t − k)〉 2 1 g(k)  √
〈ψ (t), ψ (2t − k)〉 2 corresponding frequency bands through different layers, and each frequency band has a series of wavelet packet coefficients. For the nth layer decomposition, the wavelet packet transform provides 2n different subspaces, and each subspace corresponds to a frequency band. Wavelet packet decomposition can be implemented using a series of convolutions with high-pass filters and low-pass filters. The high-pass filter h(·) and low-pass filter g(·) can be defined as Eqs 1, 2. Frontiers in Physiology (1) (2) where ϕ(t) is the scale function, ψ(t) is the wavelet function, 〈·, ·〉 represents the inner product, and t and k are variables. h(·) and g(·) satisfy Eq. 3. g(k)  (−1)k h(1 − k) 04 (3) frontiersin.org Deng et al. 10.3389/fphys.2023.1090937 FIGURE 4 The structure of LW-FHRNet. Notes: Conv2D: Convolution2D; BN: Batch Normalization; Maxpool: Max pooling; Avgpool: Average pooling. convolutional neural networks. By assigning different weights to each part of the input, more important information can be extracted to help the model make more accurate judgments without imposing greater overhead on the model’s computation and storage. Inspired by the above work, a lightweight network based on a cross-channel attention mechanism, LW-FHRNet, is proposed in this work to assist in the diagnosis of fetal distress symptoms, as shown in Figure 4. The main structure of the network contains two stages and a total of four ECA-Shuffle units. First, the feature maps based on wavelet packet coefficients are used as the input layer of the model. Subsequently, the image is conventionally convolved and the size of the output feature matrix is reduced to 1/4 of the input image using the maximum pooling operation. Then, feature extraction is performed by 4 ECA-Shuffle units to fully learn the feature unit information. Finally, regular convolution and average pooling are performed, and the output features are sent to the fully connected layer for classification. Based on the ShuffleNet-V2 units, this study constructs two types of ECA-Shuffle units by integrating the cross-channel attention module without dimensionality reduction, as shown in Figure 5. Figure 5A (Unit A) shows the first unit of each stage. The stride of the depthwise separable convolution in both the residual branch and the identity branch of the bottleneck structure is 2, and the two output feature matrices are concatenated to 2 times their depth. The ECA strategy is used at the tail of the structure. Figure 5B (Unit B) shows the second unit of each stage. The input feature matrix is divided equally into two groups. The main branch performs a depthwise separable convolution with a stride of 1, while the other branch is left unprocessed and connected to the main branch via concat, and the feature matrix depth is kept constant. The ECA strategy is also used at the end of the structure. The lower half of the ECA-Shuffle unit is the cross-channel interactive attention module without dimensionality reduction. The detailed structure is shown in Figure 6. Given the aggregated feature y ∈ RC without dimensionality reduction, channel attention can be learned by Eq. 6. The wavelet coefficients at different frequency bands and decomposition layers can be calculated iteratively by the following equation. Si+1,2j (τ )   h(k − 2τ )Si,j (k) (4) k Si+1,2j+1 (τ )   g(k − 2τ )Si,j (k) (5) k where S0,0 is the original signal of length N, Si,j (k), k  1, 2, ..., N/2i  are the wavelet coefficients in the jth subfrequency band at the ith layer decomposition, Si+1,2j (τ), τ  1, 2, ..., N/2i+1  and Si+1,2j+1 (τ), τ  1, 2, ..., N/2i+1  are the wavelet coefficients in the (2j)-th and (2j+1)-th subfrequency bands at the (i+1)-th layer decomposition, and for the ith layer decomposition j ∈ 0, 1, ..., 2i − 1. To increase the number of datasets to obtain better model effects, db1~db5 wavelet basis functions are selected for wavelet packet coefficient decomposition in this paper. Therefore, five wavelet packet coefficient matrix maps can be obtained for each data segment to enhance the dataset. Meanwhile, each wavelet packet matrix coefficient map is resized to 224*224*3 pixels as the input layer of the neural network model. The feature map construction based on wavelet packet coefficients is shown in Figure 3B. Each FHR signal segment is converted into a total of 5 feature maps based on db1~db5 wavelet bases. 2.3 LW-FHRNet network structure To meet the application of deep neural networks on embedded and mobile terminals and maintain excellent performance, lightweight network models have emerged. In particular, the lightweight models of the MobileNet series and the ShuffleNet series are the most widely used. Depthwise separable convolution, pointwise convolution, group convolution, channel shuffle and channel separation are used to reduce the number of model parameters and speed up the model computation time. Recently, the channel attention mechanism has been shown to have great potential in improving the performance of deep Frontiers in Physiology ω  σWy 05 (6) frontiersin.org Deng et al. 10.3389/fphys.2023.1090937 FIGURE 5 Detailed description of the ECA-Shuffle unit. (A) Unit A: the basic unit for spatial down sampling; (B) Unit B: the basic unit for channel split. Notes: DWConv: Depthwise separable convolution; Conv: convolution; BN: Batch Normalization; GAP: Global Average Pooling. FIGURE 6 The cross-channel interactive attention module. Notes: GAP: Global Average Pooling; C: Channel dimension; H: Height; W: Width. Frontiers in Physiology 06 frontiersin.org Deng et al. 10.3389/fphys.2023.1090937 TABLE 1 The structure parameter information of LW-FHRNet. Layer Output size Kernel size Output channel Input 224 × 224 - 3 Conv 112 × 112 3×3 MaxPool 56 × 56 3×3 Stage1 28 × 28 - 116 Stage2 14 × 14 - 232 Conv 14 × 14 1×1 1×1 14 × 14 - - AvgPool FC 24 1024 1 The normalization and ReLU, layers that follow each convolutional layer are not shown above because they do not change the output feature shape. Conv: convolutional layer; MaxPool: max pooling layer; AvgPool: average pooling layer; FC: fully connect layer; stage: ECA-Shuffle uint A+ ECA-Shuffle uint B.   log2 (C) b k  ψ (C)   +   γ γodd If the weight of yi is calculated by only considering the interaction between yi and its k neighbors and all channels share the same learning parameters, Eq. 6 can be written as Eq. 7. k ⎝wj yi ⎞ ⎠, yi ∈ Ωk ωi  σ ⎛ i j j where |t|odd represents the nearest odd number of t. To reduce the computational cost and training time, γ and b are empirically set to 2 and 1, respectively. The details of the lightweight network: LW-FHRNet structure designed in this work are shown in Table 1. The first operation of each stage is the ECA-Shuffle unit A, which realizes the doubling of feature dimensions, followed by the ECA-Shuffle unit B, which realizes the subsequent operations. The process of the fetal distress classification algorithm based on a lightweight network is described in Table 2. After preprocessing and 20-min length segmentation, the dataset is randomly divided into a training set and a testing set in proportion. Each segment is subjected to wavelet packet decomposition based on db1 to db5 wavelet basis functions to obtain five feature maps. Iterative testing of model tuning is performed with the training set data to obtain the optimal model. The testing set is subjected to category prediction under the optimal model, and the final category attribution is decided by voting on the five feature maps of each data segment. (7) j1 where Ωki indicates the set of k adjacent channels of yi . This strategy can be easily implemented by a fast 1D convolution with kernel size k, i.e., ω  σ C1Dk y (8) where C1D denotes 1D convolution. Considering each channel and its k nearest neighbors, computing local cross-channel interaction information instead of all channels effectively improves computational efficiency. This efficient channel attention calculation can be quickly implemented by 1D convolution. Thus, k is the key parameter and the size of the convolution kernel of the 1D convolution, which determines the range and convergence of the local cross-channel interaction. To avoid resource-consuming cross-validation adjustment, an adaptive method is used to select the appropriate k value. According to the properties of group convolution, the high-dimensional (lowdimensional) channels are proportional to the long-distance (shortdistance) convolution for a fixed number of groups. Similarly, the coverage of the interaction (i.e., the size k of the 1D convolution kernel) is proportional to the channel dimension C. The mapping relationship between k and C is shown in Eq. 9. C  ϕ(k) 3 Results 3.1 Dataset The database in this paper uses the publicly available dataset CTU-UHB, which comes from the Czech Technical University in Prague (CTU) and the University Hospital in Brno (UHB) (Chudacek et al., 2014). A total of 552 CTG records were collected in the database. These records were carefully selected from 9,164 records collected by UHB from 2010 to 2012. The sampling rate of CTG data is 4 Hz, and each CTG record contains FHR sequences and UC sequences. The records in the database were all singleton gestations, all gestational ages greater than 36 weeks and no known congenital developmental defects. The quality of the FHR signal was greater than 50% in every 30-min (9) Since the channel dimension is generally an exponential multiple of 2, the non-linear mapping relationship is represented by an exponential function with a base of 2. Thus, Eq. 9 can be rewritten as Eq. 10. C  ϕ(k)  2(γ*k−b) (10) Consequently, the size k of the convolution kernel can be calculated automatically based on the number of channels C, which is given by Eq. 11. Frontiers in Physiology (11) 07 frontiersin.org Deng et al. 10.3389/fphys.2023.1090937 are obtained (Ito et al., 2022). predicted fetal acidemia by calculating iPREFACE (10), iPREFACE (30) and iPREFACE (60) at 10, 30, and 60 min before delivery. The results showed that iPREFACE (30) was slightly better than iPREFACE (60) but significantly better than iPREFACE (10). To enhance the sample size, a 20-min segmentation is performed after preprocessing the 60-min data before delivery. After splitting the samples into 20-min data segments, 106 abnormal sample segments are obtained. To avoid the effect of overfitting or underfitting caused by category imbalance on the classification results, 106 samples from 512 normal samples are randomly selected. The second 20-min segment is selected to construct 106 normal sample segments for the experiment. Eighty percent of the dataset is randomly selected as the training set (85P and 85N), and the remaining 20% as the test set (21P and 21N). The wavelet packet decomposition from the db1 to db5 wavelet basis is performed separately for each FHR data segment, which constitutes 5 wavelet packet coefficient matrix feature maps. Therefore, there are 850 images in the training set and 210 images in the test set. In this paper, each 20-min segment of FHR data is subjected to wavelet packet decomposition based on db1 to db5 wavelet basis functions to obtain five wavelet coefficient matrix feature maps. Category attribution is determined by voting on the 5 feature maps. The category voting process is shown in Figure 7. First, each feature map of the segment is classified. Subsequently, the frequency of each category label is calculated for the segment. Finally, the class with higher frequency is selected as the category of this FHR segment. window. Available biochemical parameters of the umbilical artery blood sample (pH) were recorded for each sample. The pH value is a marker of blood acid-base balance and can provide information on possible fetal acidosis caused by intrauterine hypoxia. A lower pH value represents a more severe degree of fetal acidosis (Vayssiere et al., 2007). showed moderate ability to detect mild acidosis at pH ≤ 7.15 and better ability to detect more severe acidosis at pH ≤ 7.05. Therefore, in this paper, pH = 7.05 was chosen as the criterion to classify the data into two categories. Data with a pH value greater than 7.05 are considered normal, and data with a pH value less than or equal to 7.05 are considered abnormal. Based on this discriminant, 44 abnormal samples and 508 normal samples TABLE 2 Details of LW-FHRNet classification algorithm. Input: Strain training sample sets; Ltrain training label sets, Stest testing sample sets; Ltest testing label sets test Output: Prediction label L~ of the Stest 1: for dbi in [db1, db2, db3, db4, db5] do train 2: Ftrain ) # PWTdbi (·) is the wavelet packet decomposition based dbi  PWTdbi (S on the dbi wavelet basis functions train train 3: Ldbi  L test 4: Ftest ) dbi  PWTdbi (S test 5: Ltest dbi  L 6: end for 7: # training procedure 8: Initialize parameters and weights 9: for i in [1, 2, 3, 4, 5] do train 10: metrics = LW − FHRNet(Ftrain dbi , Ldbi ) 11: Train the LW-FHRNet model by optimizing the loss function 12: end for 13: return model LW-FHRNet-best 3.2 Experimental setup 14: # testing procedure 15: for i in [1, 2, 3, 4, 5] do predict ← LW-FHRNet-best (Ftest 16: Ltest dbi dbi ) 17: end for test test test test test 16: L~  vote(Ltest db1 , Ldb2 , Ldb3 , Ldb4 , Ldb5 ) #Vote () s a voting function test 17: return L~ 3.2.1 Environment The network structure proposed in this paper is trained and tested on the CTU-UHB dataset. The experimental platform is a computer equipped with an Intel Xeon(R) CPU E3-1535M v6 @ FIGURE 7 An example of the category voting process. Notes: P: Positive; N: Negative. Frontiers in Physiology 08 frontiersin.org Deng et al. 10.3389/fphys.2023.1090937 3.10 GHz x 8, Quadro P5000 GPU and 32 G RAM. The system is Ubuntu 18.04.6LTS, the development environment is TensorFlow 2.6.2, and the language used is Python. to replace group convolution to further improve the inference speed. 3.2.2 Metrics 3.3 Experiment 1: Selection of wavelet packet decomposition layers To evaluate the classification performance of the model, accuracy, precision, recall and F1-Score metrics are used in this paper. Additionally, model parameters and model size are introduced to evaluate the complexity of lightweight models. Finally, sensitivity (Se) and specificity (Sp) are used to observe the discriminatory ability of the model between abnormal and normal samples. Wavelet packet decomposition with different numbers of layers can obtain different detailed information. The sampling frequency of the raw data is 4 Hz. The ith layer is decomposed to obtain 2i frequency bands. The 2D image is constructed according to the frequency from the highest to the lowest. The frequency range of the jth frequency band is (24i (j − 1) ~ 24i j) Hz, j ∈ [1, 2i ]。 To select the best wavelet coefficient matrix feature map, this paper performs wavelet packet 1-layer to 5-layer decomposition to obtain the wavelet packet coefficient matrix maps of corresponding layers to test the classification performance. The experimental results are shown in Table 3. The accuracy of the 2-layer and 3-layer decomposition is higher, and the accuracy of the 4-layer and 5layer decomposition gradually decreases. The 2-layer decomposition achieves optimal performance with 95.24% accuracy, 100% precision, 90.48% recall and a 95.00% F1-score. Therefore, the feature map based on 2-layer wavelet packet decomposition is chosen as the input of the model in this paper. That is, the signal is decomposed into four frequency bands:0–1 Hz, 1–2 Hz, 2–3 Hz and 3–4 Hz. And the wavelet packet coefficients in the corresponding frequency bands are used to jointly construct the feature maps. 3.2.3 Baselines The commonly used lightweight networks MobileNetV3Small, MobileNetV3-Large and ShuffleNet-V2 are introduced as the baselines of this research. MobileNetV3 introduces the channel attention module based on MobileNetV2 to enhance the adaptive capability of the model by assigning different weights to different channels. MobileNetV3 has two versions: small and large. ShuffleNet-V2 proposes the concept of channel separation TABLE 3 Performance comparison of feature maps constructed by different layers of wavelet packet decomposition. Decomposition Level Accuracy (%) Precision (%) Recall (%) F1score (%) layer 1 83.33 85.00 80.95 82.93 layer 2 95.24 100 90.48 95.00 layer 3 90.48 94.74 85.71 90.00 layer 4 80.95 84.21 76.19 80.00 layer 5 76.19 76.19 76.19 76.19 3.4 Experiment 2: The effective role of local cross-channel interactive attention mechanisms The channel attention mechanism has great potential to improve the performance of deep convolutional neural networks. In this paper, we introduce a cross-channel local interaction The bold values means the best performance. FIGURE 8 Confusion matrix. (A) The proposed LW-FHRNet, (B) the proposed LW-FHRNet without the ECA module. Frontiers in Physiology 09 frontiersin.org Deng et al. 10.3389/fphys.2023.1090937 performs poorly in terms of accuracy, with only 83.33%. Due to the low number of parameters in the ShuffleNet-V2 model, its performance is relatively poor, with an accuracy of 83.33%. LW-FHRNet incorporates an efficient cross-channel attention mechanism without downscaling on the base unit of ShuffleNet-V2. The channel interaction strategy effectively improves the performance of channel attention and enables LW-FHRNet to have a more accurate recognition performance. The ROC curves of LW-FHRNet and other commonly used lightweight network models are shown in Figure 9A. The proposed network in this paper has the best performance with 97.96% AUC. A comparison of the accuracy and model size of LW-FHRNet with other commonly used lightweight networks for fetal distress classification is shown in Figure 9B. LW-FHRNet achieves 95.24% accuracy for fetal distress classification, which is higher than other commonly used lightweight networks. Additionally, it has the lowest computational cost, and the number of network parameters is only 0.33 M, which is much lower than other commonly used lightweight networks. attention strategy without dimensionality reduction to improve the performance of lightweight models. Experiments are conducted on the dataset of this paper using a lightweight network with and without an ECA module. The confusion matrix of whether the proposed lightweight model contains ECA modules is shown in Figure 8. Table 4 shows the model performance comparison with and without the ECA module. The lightweight model accuracy with the ECA module is as high as 95.24%, and the accuracy of the lightweight model without the ECA module is 92.86%. The experimental results show that the lightweight model with the ECA module improves performance in fetal distress classification. 3.5 Experiment 3: Lightweight model comparison experiment To clarify the performance of the network, this paper performs a comparative test with different lightweight networks. The classification performance of fetal distress under different lightweight networks is measured using accuracy, precision, recall, F1-score and model size metrics. The test performance comparison of the LW-FHRNet network with other commonly used lightweight networks is shown in Table 5. MobileNetV3 improves MobileNetV2 by using a deep separable convolution +SE channel attention mechanism + residual structure connection to further reduce the computational effort. The overall structure of small and large is the same, and the difference is the number of bnecks and channels. MobileNetV3-Small achieves 85.71% accuracy, proving that the network has a strong feature learning capability. MobileNetV3-Large has better accuracy than MobileNetV3-Small, but the number of network parameters has increased significantly due to the increase in the number of bnecks and channels. The ShuffleNet-V2 network improves the ShuffleNet-V1 network architecture in terms of optimizing memory access cost (MAC), reducing network fragmentation, and decreasing element operations. Due to the small number of parameters in the ShuffleNet-V2 model, it 4 Discussion In this paper, a lightweight network based on cross-channel interactive attention mechanism is proposed to effectively fuse channel features and reduce model complexity to help obstetricians to objectively assess fetal distress. In the experiments, the classification effects of wavelet packet decomposition with different layers as feature maps were first compared. And the optimal number of wavelet packet decomposition layers was chosen as 2-layer. Then two different network architectures (LW-FHRNet and LW-FHRNet-without-eca) were used. The results showed that the attention machine module effectively improves the classification performance of fetal distress. Finally, a comparison with other lightweight models was made to show that the lightweight network proposed in this paper outperforms other common lightweight networks. To analyze the significance of the results, the algorithm in this paper is compared with recent related work in the diagnosis of fetal distress using the CTU-UHB database. The results are shown in Table 6, which measures the performance of this research work in terms of accuracy (Acc), sensitivity (Se) and specificity (Sp). Compared with (Zarmehri et al., 2019), the method of this paper has higher Se and Sp under the same fetal distress division criteria, which further highlights the advantages of our model. Compared with (Alsaggaf et al., 2020), they also have good classification accuracy, but they use the traditional machine learning TABLE 4 Lightweight model performance comparison with and without the ECA module. Model (%) Accuracy (%) Precision (%) Recall (%) F1 score (%) LW-FHRNet 95.24 100 90.48 95.00 LW-FHRNetwithout-eca 92.86 100 85.71 92.31 The bold values means the best performance. TABLE 5 Performance comparison of different lightweight models for fetal distress classification. Network Accuracy (%) Precision (%) Recall (%) F1 score (%) Parameter (M) Model size (M) MobileNetV3-Small 85.71 82.61 90.48 86.36 1.53 5.84 MobileNetV3-Large 90.48 94.74 85.71 90.00 4.23 16.13 ShuffleNet-V2 83.33 85.00 80.95 82.93 1.27 4.85 LW-FHRNet(ours) 95.24 100 90.48 95.00 0.33 1.27 The bold values means the best performance. Frontiers in Physiology 10 frontiersin.org Deng et al. 10.3389/fphys.2023.1090937 FIGURE 9 Classification performance of different lightweight models. (A) ROC curves of different lightweight models; (B) Acc and parameters of different lightweight models, where green, blue, purple, and red refer to MobileNetV3-Small, MobileNetV3-Lagre, ShuffleNetV2, and LW-FHRNet (Ours). TABLE 6 Comparison of recent studies on the prediction of fetal distress using the CTU-UHB database. Author Division criteria Method Performance (%) Acc Se Sp Comert and Kocamaz. (2018) pH ≤ 7.15 BFS, DWT + SVM 67.00 57.42 70.11 Fuentealba et al. (2019) PH<7.05; BDecf≥12pH>7.20; BDecf≥12 CEEMDAN, TV-AR + SVM 81.7 79.5 86.45 Zarmehri et al. (2019) pH ≤ 7.05 FFT — 63.60 80.10 Alsaggaf et al. (2020) pH < 7.15 Morphological, linear, non-linear, CSP + SVM 94.75 74.29 99.55 Zeng et al. (2021) pH ≤ 7.05; BE ≤ −10 CWT, WTC, XWT + ECSVM 67.2 85.2 66.1 Liu et al. (2021) pH ≤ 7.15 CNN-BiLSTM + Attention, DWT 71.71 75.23 70.82 Baghel et al. (2022) pH ≤ 7.15 1D CNN 99.09 — — Ours pH ≤ 7.05 WPT + LW-FHRNet 95.24 90.48 100 BFS: basic feature set; DWT: discrete wavelet transform; CEEMDAN: complete ensemble empirical mode decomposition with adaptive noise; TV-AR: time-varying autoregressive; CSP: common spatial pattern; CWT: continuous wavelet transform; WTC: wavelet coherence; XWT: Cross-wavelet Transform; ECSVM: ensemble cost sensitive SVM; WPT: wavelet packet transform; Acc: Accuracy; Se: Sensitivity; Sp: Specificity. The bold values means the best performance. pH as a criterion for classification, since pH is an objective response to the fetal oxygen cell supply (Zarmehri et al., 2019) and also to the severity of fetal acidosis (Vayssiere et al., 2007). However, as shown in Table 6, a variety of pH values were used in different research works. There is not yet a universally accepted pH value. In future research work, the study will focus on exploring the pH value of pathological samples. Meanwhile, the BDecf index can reflect the degree of fetal acidosis (Liu et al., 2021). Therefore, a more precise classification of fetal distress can be performed by combining pH and BDecf in subsequent studies. classification method, which requires manual design to extract a large number of features. The feature extraction process is complex and computationally intensive. Compared with (Baghel et al., 2022), they have higher accuracy than the model in this paper, but they use regular CNN convolution for feature extraction. The parameter number and computational time still need to be improved and optimized for end-application deployment. In conclusion, the lightweight network model based on the cross-channel interactive attention mechanism proposed in this paper achieves better classification results in fetal distress diagnosis. The ShuffleNet-V2 unit combined with the local crosschannel interactive attention mechanism is used to build a lightweight network, which ensures a low number of parameters and achieves effective network performance improvement. However, one limitation of the study in this paper is the criteria for discriminating between normal and distressed samples. The current work generally endorses the use of umbilical artery blood Frontiers in Physiology 5 Conclusion In this work, a lightweight network (LW-FHRNet) based on ECA-Shuffle units is proposed for fetal distress classification of FHR signals. After preprocessing, the FHR signal is segmented into 20- 11 frontiersin.org Deng et al. 10.3389/fphys.2023.1090937 Author contributions min segments, and the wavelet packet decomposition operation based on db1 to db5 wavelet basis functions is performed on each segment. Each segment obtains five wavelet packet coefficient matrix feature maps, which are used as input to the model and vote on the classification result. The ECA-Shuffle unit performs feature extraction on the feature map to fully learn the feature information. We integrate an efficient local cross-channel interactive attention mechanism without dimensionality reduction to reduce model complexity and ensure performance improvement. In this paper, the CTU-UHB open source database is used to test the classification performance of the proposed network. A pH value of 7.05 was used as the gold standard for classification. The proposed algorithmic model achieves excellent results of 95.24%, 90.48%, and 100% for Acc, Se and Sp, respectively. Although the proposed lightweight network achieved good results in classifying fetal distress, there is still a gap to reach the clinical diagnosis level of physicians. In order to achieve better auxiliary diagnosis, we will do further exploration in future work. On the one hand, the data from clinical fetal heart monitoring contain simultaneous UC signals and FHR signals, but only FHR signals are used to assess fetal distress because of the poor quality of UC signals in publicly available datasets. In the clinic, the UC signal is also an important basis for physicians to diagnose fetal distress. Therefore, the combination of FHR signals and UC signals needs to be considered in further studies. On the other hand, we are considering more time-frequency transform features to improve the classification performance for fetal distress, including Empirical Wavelet Transform, Hilbert-Huang Transform, Singular Spectrum Analysis, etc. YD, ZZ, and PJ contributed to the conception and design of the study. All authors contributed to the interpretation of the results. All authors provided critical feedback and helped shape the research, analysis, and manuscript. Funding This work was supported by the National Natural Science Foundation of China (Grant No.62071162). 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. 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. Supplementary material Data availability statement The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys.2023.1090937/ full#supplementary-material Publicly available datasets were analyzed in this study. 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ORIGINAL RESEARCH published: 05 September 2018 doi: 10.3389/fpubh.2018.00254 Exit-Knowledge of Ambulatory Patients About Medications Dispensed in Government Hospital in Eastern Ethiopia: The Need for Focused Patient Counseling Nigatu Hirko 1 , Dumessa Edessa 2 and Mekonnen Sisay 3* 1 Department of Pharmacy, Bisidimo General Hospital, Oromia, Ethiopia, 2 Department of Clinical Pharmacy, School of Pharmacy, College of Health and Medical Sciences, Haramaya University, Harar, Ethiopia, 3 Department of Pharmacology and Toxicology, School of Pharmacy, College of Health and Medical Sciences, Haramaya University, Harar, Ethiopia Edited by: Sunjoo Kang, Cheju Halla University, South Korea Reviewed by: Anton Civit, Universidad de Sevilla, Spain Iffat Elbarazi, United Arab Emirates University, United Arab Emirates *Correspondence: Mekonnen Sisay mekonnensisay27@yahoo.com Specialty section: This article was submitted to Public Health Education and Promotion, a section of the journal Frontiers in Public Health Received: 18 June 2018 Accepted: 16 August 2018 Published: 05 September 2018 Citation: Hirko N, Edessa D and Sisay M (2018) Exit-Knowledge of Ambulatory Patients About Medications Dispensed in Government Hospital in Eastern Ethiopia: The Need for Focused Patient Counseling. Front. Public Health 6:254. doi: 10.3389/fpubh.2018.00254 Background: In the counseling process, a multi-cultural competence of dispenser is among the key factors affecting his/her successful communication with patients for achieving optimal use of medications. For patients to use dispensed drugs appropriately, it is a must for them to understand the medication related information provided by the dispenser. Hence, the objective of this study was to identify parameters that likely affect ambulatory patients’ knowledge of medication(s) provided at the exit of outpatient pharmacy of Federal Harar Police Hospital, Eastern Ethiopia. Methods: Cross-sectional study design was employed to conduct this study. An interview of patients was made at the exit of hospital pharmacy using a semi-structured questionnaire. The interview tool primarily assessed the knowledge of the patients for a maximum of three medications provided. Statistical Package for Social Sciences (SPSS), Version 20.0, was employed for analysis of the data. Chi-squared test was done to retain parameters with potential to have association; and the retained parameters were adjusted by performing bivariate and multivariate logistic regression analyses. Results: The result showed that only 37.2, 33.4 and 28.7% of the patients were able to recall the name of the drug(s), common side effects, and actions to be taken for missed doses, respectively. The likelihood of patients’ knowledge for dispensed medications was high among patients aged 19–39 years (adjusted odds ratio [AOR]: 5.0; 95% CI: 1.04–24.2) and who thought their communication with dispenser had been polite (AOR: 4.62; 95% CI: 1.48–14.4). However, the knowledge status was found low among patients who were Afan Oromo speakers (AOR: 0.58; 95% CI: 0.35–0.95) and who came from rural residence (AOR: 0.48; 95% CI: 0.25–0.90). Conclusion: A high proportion of patients were unable to recall the drug (s) name, associated common toxicities, and actions to be taken in case of missed dose. In addition, patients who were at early adulthood and who were positive for the politeness of dispenser had better exit-knowledge of their medication. Therefore, for the patients’ clear Frontiers in Public Health | www.frontiersin.org 1 September 2018 | Volume 6 | Article 254 Hirko et al. Patients’ Knowledge About Dispensed Medication understanding of medications provided, it is mandatory to optimize patient–dispenser communication possibly by adapting multi-cultural communication skills and by providing focused training for dispensers to address factors that likely affect patient-dispenser interactions. Keywords: dispensed medications, patient, exit-knowledge, outpatient pharmacy, Federal Harar Police Hospital INTRODUCTION patients with specific conditions (e.g., pregnancy, renal, and hepatic dysfunctions); patients taking multiple medications prescribed by the same or different professionals; patients with multiple health problems; patients who are passive about their own health; patients with dementia (memory problems) and patients who have communication difficulty, young children, and patients who do not speak the same language as the dispenser, are particularly vulnerable to medication errors (6, 11). Studies that assess the exit-knowledge status of patients toward their dispensed medication at outpatient setting are very limited in Ethiopia. Therefore, this study aimed to identify dispenser and patient related factors that likely affect the knowledge of ambulatory patients toward medication dispensed at outpatient pharmacy of Federal Harar Police Hospital (FHPH). Drug dispensing is a process that ends when a patient leaves drug retail outlets with a defined quantity of medication(s) and adequate instruction (s) provided on how to use them (1). It is one of the focal areas that drug use can be promoted in healthcare system. The way in which drugs are used by the patients is commonly affected by the overall protocol of dispensers and information delivered by them during the dispensing process. The time that the dispenser spends with patients during drug use process is referred to as dispensing or counseling time which is an important indicator of the quality of service delivery (2–4); and this has to be critically considered with regard to the dispenser’s role and competence in effective patient counseling. Pharmacists should be engaged in optimizing patient safety throughout the medication use processes. The role of the dispensers have been proven to improve several health outcomes including greater patient safety, better disease and drug therapy management, more efficient healthcare expenditure, higher adherence status, and better quality of life (5). Therefore, patient counseling by dispenser is a key competency element in the medication treatment process. To this end, it is critical for the dispensers to provide desirable and understandable information to patients about their dispensed medications. The dispenser is in a critical position to answer whatever concerns and enquiries of patients toward their medications and even alternate therapeutic approaches they may seek or hear from others. Counseling should include an evaluation of whether the information was received as required and that the patient comprehends how to use the information he/she receives to improve positive therapeutic outcomes. Appropriate means of delivering information to patients is also essential to encourage effective and safe use of medication(s) by them. Accordingly, the Australian Pharmaceutical Advisory Council outlines the type of information and resources that should be delivered to patients. Moreover, list of medicines provided on exit from the healthcare facility should be prepared in communication and collaboration with the patient for ease of improving adherence to treatment regimens and patient outcomes (6–9). Communication failure between patients and healthcare providers can lead to gaps in the continuity of the treatment process. Health professionals are prescribing and dispensing a large number of medications without providing sufficient information to the patients. To this end, studies showed that higher workload of dispensers, defined as the number of prescriptions dispensed per dispenser working hour led to increased risk of dispensing a potentially unsafe medication and unlikely to provide thorough patient counseling about their dispensed medications (6, 10). In addition, certain patients are potentially vulnerable to medication errors. These include Frontiers in Public Health | www.frontiersin.org METHODS Study Area, Design, and Period This study was conducted at FHPH outpatient pharmacy. The FHPH is one of the government hospitals found in Harar town located 526 km away from the capital of Ethiopia, Addis Ababa to the east. An institution-based cross-sectional study design was employed to assess the knowledge status of patients served at the outpatient pharmacy of the hospital from February to April, 2016. Study Population and Inclusion/Exclusion Criteria Ambulatory patients who visited the pharmacy unit of FHPH to receive medications dispensed to them during the data collection period were included for the study. Those who were seriously ill and/or who could not give their consent to participate in the study were excluded. In addition, due to concerns pertaining to patients’ retention and recall capability of information in case of high number of dispensed drugs, those who received four and more drugs from the pharmacy were not considered for the interview. Sample Size Determination and Sampling Technique The number of study participants was determined by using single population proportion formula (Z2α pq/d2 ). For this, alpha (α) value of 0.05, confidence level (Cl) of 95% and proportion (p) of 0.50 for knowledge status of patients at the exit of outpatient pharmacy, and 5% margin of error (d) were employed. Accordingly, 384 patients were obtained as the exact sample size of study population was not known in such prospective study and this number was finally adjusted to 422 patients by adding 10% for certain methodological non-contingency. During the time of 2 September 2018 | Volume 6 | Article 254 Hirko et al. Patients’ Knowledge About Dispensed Medication Statistical Analysis data acquisition, convenient sampling technique was employed to enroll study participants for interview. The data were coded for suitable entry into Epi-info Version 3.5.1 and exported to Statistical Package for Social Science version 20.0 (IBM Statistics, Armonk NY, United States) for statistical analysis. Socio-demographics characteristics, patientdispenser communication and interaction parameters, and exitknowledge level of patients about the dispensed medication(s) were summarized by using frequency and percentages (univariate analyses). Potential variables with p < 0.2 by Pearson chi-squared test (χ2 ) were retained for subsequent consideration in binary logistic regression analysis. Finally, variables retained after the χ2 test were regressed against the knowledge status of patients. Accordingly, age, place of residence, primary language spoken by the patient, frequency of pharmacy visit within the last 6 months, patient’s perception about dispenser’s politeness during counseling process, and perceived interaction status between patient and dispenser were the key variables considered for both bivariate and multivariate regression against the outcome variable. Significant association was declared at p < 0.05 and 95% confidence level. Study Variables Socio-demographic parameters such as age, sex, area of residence, educational status, ethnicity, and primary language of communication along with patient-dispenser communication parameters were considered as explanatory variables. In addition, patient’s level of exit-knowledge about their dispensed medication was referred as a key outcome variable. A patient was assumed to be with adequate exit-knowledge about the medication(s) dispensed when he/she definitely addressed two-thirds and more of the necessary knowledge related questions. Data Collection Tools and Methods A customized semi-structured data collection tool was employed for interviewing the patients during exit from the hospital pharmacy. Good dispensing practice variables adopted by the World Health Organization (WHO) and the Ethiopian Food, Medicine and Healthcare Administration and Control Authority (FMHACA) were considered partly for the preparation of the questionnaire (12, 13). This tool consisted of patient related characteristics including age, sex, place of residence, educational level, occupation, marital status, and type of language spoken, along with patient-dispensary communication and interaction parameters that likely affect the knowledge level of how to take dispensed medications. Accordingly, the tool has two sections: the first section contained the socio-demographic and patient-dispenser characteristics; the second part of the tool addressed the knowledge status of patients about the medication(s) dispensed to them at exit of hospital pharmacy (Supplementary Table 1). Ethical Consideration Study approval and ethical clearance was sought and received from Haramaya University, College of Health and Medical Sciences, School of Pharmacy. A clearance letter was also obtained from the College of Health and Medical Sciences with reference number C/A/R/D/01/1611/16 for conducting this research in FHPH. Official permission was then received from hospital administrator to start the study. Voluntary, informed, written and signed consent was also obtained from every study participant after the purpose of the study was introduced to him/her prior to conducting actual interview. Confidentiality of the collected data was maintained in such a way that the data collection tool was kept anonymous. Pretest and Quality Control RESULTS In this study, various techniques were tried to guarantee the quality of the data collected. Before the actual data collection, the drafted interview tool was ultimately enhanced by pre-testing on 5% of patients (22 patients) at dispensary unit of Jugel Hospital which is found in the same town. Following certain amendments based on the pretest data, the data collection tool was employed for the actual data gathering. The data collection was conducted by two pharmacists who had been vividly trained about the purpose and method of data collection. The data collectors handled the interview process while also checking the responses provided immediately after completing every interview. Consistency of the questionnaire was maintained by translating English language into local languages (Afan Oromo and Amharic). These local language contents of the tool were back translated into English to ensure consistency of the translations. At every interview, each patient was also well informed about the goal of the study while simultaneously addressing the importance of replying to all the inquiries sincerely as well. Finally, the completeness of each datum was ensured daily by the investigators and comments were provided to the data collectors accordingly. A total of 422 patients were included in the study. More than half of the patients were in age range between years 19 and 39 (57.8%), were married (70.6%), were Orthodox Christian (48.6%), had finished secondary levels of education (41.2%), belonged to the Oromo (41.9%) and Amhara (37.2%) ethnic groups, were Amharic language speakers (65.9%) and were urban dwellers (83.9%). Similarly, about three-fourth (74.2%) and two-thirds (65.6%) of the patients were more frequent visitors of pharmacy and were comfortable with the waiting facility of the dispensary unit, respectively. Moreover, at least two-thirds of the patients perceived about their interaction with dispensers as promising. Accordingly, the patients thought that their interaction with dispensers was encouraging in terms of ascent of dispensers (87.2%), manner of dispensers (82%), guidance of dispensers (79.4%), and communication of dispensers (68.2%) (Table 1). At exit from the dispensary unit, study patients were assessed whether they had the required information of the medication they received as summarized in Table 2. As per the result, at least more than half of the patients recalled the required information related to their the medication frequency of use (82.9%), route Frontiers in Public Health | www.frontiersin.org 3 September 2018 | Volume 6 | Article 254 Hirko et al. Patients’ Knowledge About Dispensed Medication TABLE 1 | Socio-demographics and perceived communication status of study participants at FHPH, Harar, February-April, 2016. TABLE 1 | Continued Characteristics Characteristics Frequency (%) Frequency (%) Oromo Sex 118 (28.0) Adarigna 19 (4.5) 7 (1.7) Male 215 (50.9) Tigirigna Female 207 (49.1) Perceived communication ranked by the patient Age (years) ≤18 13 (3.1) Poor 30 (7.1) Fair 104 (24.6) 288 (68.2) 19–39 244 (57.8) Good 40–59 126 (29.9) Perceived ascent status of the dispenser ≥60 39 (9.2) Marital status Clear 368 (87.2) Not clear 54 (12.8) Single 90 (21.3) Perceived comfort of waiting area Married 298 (70.6) Not suitable Divorced 13 (3.1) Fairly suitable 72 (17.1) Widowed 19 (4.5) Suitable 277 (65.6) Separated 2 (0.5) Perceived politeness of the dispenser Religion 73 (17.3) Impolite 27 (6.4) Muslim 148 (35.1) Fairly polite 49 (11.6) Orthodox 205 (48.6) Polite 346 (82.0) Protestant 68 (16.1) Perceived clarity of the dispenser’s guidance Catholic 1 (0.2) Occupation Farmer 5 (1.2) Not clear 39 (9.2) Fairly clear 48 (11.4) Clear 335 (79.4) Government employee 200 (47.4) Perceived sufficiency of the dispenser’ information Merchant 56 (13.3) Not enough Laborer 14 (3.3) Don’t know Private employee 28 (6.6) Enough Student 43 (10.2) Housewife 48 (11.4) Retirement 28 (6.6) Can read and write of administration (82.5%), direction of use (72.3%), expected therapeutic outcome (63%), storage conditions (54.9%), and drug interaction (54.7%). Contrary to these, only 37.2, 33.4 and 28.7% of the patients were able to remember name of the medication, potential toxicities, and actions taken in cases of forgotten doses, respectively. Generally, only 38.6% of the patients fulfilled the predefined criteria and hence considered to have adequate knowledge (Table 2). On binary logistic regression analysis, several variables were found to have association with patients’ knowledge of how to use their dispensed drugs (Table 3). Accordingly, patients who thought the manner of dispenser as fairly positive (crude odds ratio [COR], 4.31; 95% CI, 1.29–14.36) or positive (COR, 3.81; 95% CI, 1.29–11.27) had more understanding of the drug (s) they received from pharmacy. Multivariate analysis also revealed a similar finding to the above bivariate analysis and patients who thought manner of the dispenser as positive had more odds of understanding for dispensed drug (s) (adjusted odds ratio [AOR], 4.62; 95% CI, 1.48–14.4) compared to patients who thought manner of the dispenser as negative. Furthermore, patients who were in the age range between years 19 and 39 (AOR: 5.0; 95% CI: 1.04–24.2) had more likelihood of understanding of the drug(s) they received compared to patients aged ≤ 18 years. Nevertheless, a significantly reduced exit knowledge about the 25 (5.9) 32 (7.6) Primary 121 (28.7) Secondary 174 (41.2) Tertiary 70 (16.6) Ethnicity Oromo 177 (41.9) Harari 44 (10.9) Amhara 157 (37.2) Tigrie 27 (6.4) Somali 10 (2.4) Other* 7 (1.7) Place of residence Urban 354 (83.9) Rural 68 (16.1) Frequency of dispensary unit visit in the last 6 months First time 24 (5.7) Second time 85 (20.1) Repeated times 313 (74.2) Primary language of communication by patient Amharic 278 (65.9) (Continued) Frontiers in Public Health | www.frontiersin.org 34 (8.1) 273 (64.7) Asterisk (*) stands for Gurage, Sidama, and Argoba. FHPH, Federal Harar Police Hospital. Educational status Illiterate 115 (27.3) 4 September 2018 | Volume 6 | Article 254 Hirko et al. Patients’ Knowledge About Dispensed Medication can be affected by a multitude of factors. Among which, this study mainly emphasized on patient and dispenser related factors that may possibly affect the exit-knowledge of dispensed drugs. Binary logistic regression was applied to show whether there is a statistically significant association between the exit- knowledge of patients with several predictor variables including residence of patients, age, the type of language they primarily spoke, perceived politeness of the dispensers, frequency of hospital pharmacy visits and the nature of dispenser-patient interactions. In this study, 89.2, 82.5, 72.3, 63, 54.9, and 54.7% of patients recalled frequency of medication, route of administration, medication instruction, expected therapeutic outcome, proper storage conditions, and drug interactions, respectively. However, less than half of the patients (37.2, 33.4, and 28.7%) were able to recall the name of the medication, the major side effects, and actions taken in cases of missed doses, respectively. Overall, less than half (38.6%) of the ambulatory patients met the predefined criteria of exit knowledge about dispensed drugs. In relation with this finding, study conducted at rural Gambia showed that following an interview on exit knowledge of dispensed drugs, 60.4, 5.4, and 17.2% patients correctly recalled the drug dosage, the duration of treatment, and the purpose of treatment, respectively. Overall, 16.1% of patients’ responses met the predefined criteria for ‘good’ level of exit knowledge of dispensed drugs (14). Similarly, a research conducted at Jimma University Specialized Hospital showed that the mean patients’ exit knowledge score was found to be 3.7 out of 7. Routes of administration (100.0%), dose (96.1%), frequency (95.5%), indication (89.3%), duration of therapy (49.6%), and the name of medicine (15.1%) were recalled among the dispensed drugs (15). In this study, the multivariate analysis indicated that statistically significant exit knowledge of dispensed drugs was observed in patients whose age was within 19–39 years compared to patients aged ≤18 years [AOR = 5.0; 95% CI: 1.04–24.2]. In agreement to our study, a study conducted in outpatient settings revealed that being adult and older age were associated with higher levels of exit knowledge compared to younger patients (p < 0.05) (16). Furthermore, another study indicated that older patients were generally more satisfied with the consultations given by dispensers, having greater exit knowledge of dispensed drugs as well (14, 17). On the other end, a contradictory finding was also reported by Marks et al. (18) in which the medication knowledge score was affected positively by younger age. Besides, a study done by Kerzman et al. (19) has also reported another findings stating that there was no statistically significant effect of age on medication knowledge of patients. Residence of patients can also affect the exit knowledge of ambulatory patients about their dispensed medication(s). The knowledge of dispensed drugs was significantly decreased among patients who were rural residents compared to those who were urban dwellers. In concordant to the present finding, a study conducted at Shambu primary hospital, southwest Ethiopia indicated that misunderstanding of dosage regimen instructions was significantly associated with residence. Those patients who came from rural residence were more likely to misunderstand instructions compared to urban dwellers (χ2 = 13.8, p < 0.001) (20). TABLE 2 | Knowledge status of patients about their dispensed drugs at exit of pharmacy of FHPH, February-April, 2016 (n = 422). Patient’s knowledge status (recalling capability) Frequency (%) Name of medication(s) dispensed No 265 (62.8) Yes 157 (37.2) Medication’s indication No 155 (36.7) Yes 267 (63.3) Route of administration No 74 (17.5) Yes 348 (82.5) Duration of therapy No 199 (47.2) Yes 223 (52.8) Medication’s frequency No 72 (17.1) Yes 350 (82.9) Drug interaction No 191 (45.3) Yes 231 (54.7) Common potential toxicities No 281 (66.6) Yes 141 (33.4) Direction on how to use medication No 117 (27.7) Yes 305 (72.3) Actions taken for forgotten dosage No 301 (71.3) Yes 121 (28.7) Appropriate handling of the received drug No 191 (45.4) Yes 231 (54.7) Medication’s label No 138 (32.7) Yes 284 (67.3) Expected therapeutic outcome No 156 (37.0) Yes 284 (63.0) Average sufficiency of the awareness Sufficient (at least two-thirds of correct answers) 163 (38.6) Not sufficient (less than two-thirds of correct answers) 259 (61.4) FHPH, Federal Harar Police Hospital. dispensed drug(s) was noted among patients who spoke Afan Oromo (AOR: 0.58; 95% CI: 0.35–0.95) and who were rural residents (AOR, 0.48; 95% CI, 0.25–0.90) compared to patients who spoke Amharic and who were urban residents, respectively (Table 3). DISCUSSION This study generally included 422 patient attendees in the FHPH during the study period. The overall exit-knowledge of patients Frontiers in Public Health | www.frontiersin.org 5 September 2018 | Volume 6 | Article 254 Hirko et al. Patients’ Knowledge About Dispensed Medication TABLE 3 | Regression analysis for factors associated with knowledge of patients for dispensed drugs at dispensary unit of FHPH, Harar, February-April, 2016. Variable COR [95% CI] P-values AOR [95% CI] P-values Age (years) 1 ≤18 1 19–39 4.2 [0.92–19.46] 0.065 5.0 [1.04–24.2] 0.045* 40–59 2.2 [0.46–10.42] 0.32 2.24 [0.45–11.1] 0.32 ≥60 5.2 [1.02–26.72] 0.047 4.36 [0.84–23.8] 0.08 0.006 0.48 [0.25–0.90] Residence Urban 1 Rural 0.43 [0.24–0.80] 1 0.02* Primary language the patient spoke Amharic 1 Oromo 0.60 [0.38–0.95] 0.03 0.58 [0.35–0.95] 1 0.03* Adarigna 1.26 [0.49–3.19] 0.63 1.12 [0.41–3.02] 0.82 Tigirigna 0.93 [0.15–5.66] 0.94 0.89 [0.13–5.97] 0.90 Perceived politeness of the dispenser Negative 1 1 Fairly positive 4.31 [1.29–14.36] 0.017 1.71 [0.68–4.32] 0.55 Positive 3.81 [1.29–11.27] 0.015 4.62 [1.48–14.4] 0.008* Frequency of dispensary visit in the last 6 months First time 1 1 Second time 2.0 [0.72–5.55] 0.18 2.36 [0.78–7.08] 0.12 Repeated visit 1.94 [0.75–5.03] 0.17 1.70 [0.61–4.71] 0.30 Perceived interaction of patient with dispenser Poor 1 Fair 1.52 [0.61–3.75] 0.36 1.38 [0.52–3.68] 1 0.52 Good 1.90 [0.82–4.43] 0.13 1.71 [0.68–4.32] 0.25 Asterisk (*) shows statistical significance. CI, confidence interval; FHPH, Federal Harar Police Hospital. good communication also benefits the healthcare system as a whole by making it more efficient and cost-effective (27). Looking at perceived interaction status of patients with pharmacist, in spite of statistically insignificant association, there is an increasing trends of exit knowledge level of ambulatory patients when the perceived interaction become stronger. Accordingly, patients who perceived their interaction as good and fair have 1.71 and 1.38 times more odds of exit knowledge of dispensed drugs, respectively, compared to those who perceived the patient-pharmacist interaction as poor. Supporting this study, another study showed that there was a close correlation between patients’ knowledge of dispensed drugs and pharmacist interaction (r = 0.95) with the patient. The interaction between dispensers and patients increases exit knowledge of dispensed medicines and it was reported that written directions are very useful when they are supplemented by an oral explanation. The most effective tasks in promoting the pharmacist–patient interaction were obtaining patients’ history and provision of verbal instruction. Besides, eye contact was significantly related to patient perceptions of clinician empathy and connectedness (28–30). However, very short dispensing time was considered as a major factor for inadequate provision of medication counseling and hence results in poor exit knowledge of patients about their dispensed drugs (31). On the top of this, dispensers can undergo interactive communication and provision of instructions by actively involving the patients in their own health. Since patients Regarding the barrier for effective communication between pharmacists and patients in outpatient settings, language barrier is one of the most important but often overlooked barriers in area of clinical settings. In the present study, the knowledge of dispensed drugs was significantly decreased among patients who were Afan Oromo speakers (AOR: 0.58; 95% CI: 0.35– 0.95) compared to those patients who were Amharic speakers. There are several literatures that stand for supporting the present finding. Generally, communication has been recognized as a huge potential barrier in healthcare provider- patient interaction and there is evidence of interference from psychosocial, cultural and linguistic barriers. The way and the language in which the healthcare provider communicates and the patient understands are very critical since poor communication may lead to nonadherence to medicines (21). Some patients may show motivation to adhere with the prescribed medications; however, fail to do so because of misunderstanding associated with several factors including low literacy level and language barriers which are essential factors for unintentional non-adherence (22). Language barriers between pharmacist-patient communications may lead to diminished patient satisfaction with treatment; lower understanding of medication counseling; reduced adherence to prescribed medications; fewer follow-up visits; and poorer treatment outcomes. (23–26). Similarly, good communication facilitates the counseling process and results in more appropriate treatment regimens and better patient compliance. Moreover, Frontiers in Public Health | www.frontiersin.org 6 September 2018 | Volume 6 | Article 254 Hirko et al. Patients’ Knowledge About Dispensed Medication thought to be affected by the number of drugs dispensed to them. On top of these limitations, patients who responded at least two-thirds of the knowledge questions were considered knowledgeable for the dispensed drugs, and this might have also resulted into underestimation of the exit knowledge status. Hence, any interpretation of the findings in this study should be done in consideration of the aforementioned limitations. forget more than half of the information delivered to them via oral communication immediately after they hear it, strategies aimed at improving patients’ recall of medical instructions must be used to assist patients with their prescribed medications (22). Another study also emphasized that pharmacists still have a way to go to fully address patients’ healthcare demands, particularly in culturally diverse settings (32–34). One report indicated that among patients with chronic conditions who came for medication refills, 41.8% believed that, although they needed counseling, it was not provided by the pharmacist (17). Coming to the perceived politeness of pharmacist, multivariate analyses revealed a statistically significant association between the politeness of pharmacist and patients’ knowledge about dispensed drugs. That is, patients who perceived the behavior of pharmacist as polite had 4.62 times more odds of the exit knowledge of dispensed drugs compared to those who perceived the pharmacist behavior as impolite. On this side, there have been some research findings and hypothesis supporting the present study. It is important that dispensers establish a positive, supportive and trusting relationship with the patient. One must adopt a friendly rather than a business-like attitude toward the patients he/she serves (35). Furthermore, the existence of optimum and positive interaction that can be achieved between pharmacists and their patients has been shown to originate from the pharmacists’ communication skill and use of motivational conversation (36, 37). Regarding the frequency of outpatient pharmacy visits, despite statistically insignificant association, patients who had experiences of second or repeated visits to the hospital pharmacy had increased odds of the exit knowledge of dispensed drugs. Consistent to the present finding, previous counseling was positively linked to the medication knowledge (P < 0.05). About 87.8–97.6% of patients who received the previous counseling showed good to excellent recognition of medication knowledge of their indications (16). CONCLUSION The present study found that less than half of the patients met the defined criteria for adequate exit knowledge and various factors related to patient and dispenser affected the exit knowledge of patients on how to use dispensed drugs. The exit knowledge was significantly increased among patients at early adulthood. Perceived politeness of dispensers by patients was also found to have statistically significant association with the exit knowledge. However, the exit knowledge was significantly decreased among patients who were rural residents and were Afan Oromo speakers. Therefore, increasing educational coverage and standards targeting the rural community and improving multicultural communication skill based trainings for dispensers will likely optimize patient–dispenser communication, which in turn will improve the exit knowledge of patients about medications dispensed to them. AUTHOR CONTRIBUTIONS NH has conceived the study, participated in study design, data acquisition, and interpretation of results. DE has also conceived the study, participated in the study design, data acquisition, statistical analysis, and interpretation of results. MS has also participated in the study design, data acquisition, analysis, and interpretation of findings, and manuscript drafting and writing. The three authors have participated sufficiently in contributing to the writing of the manuscript and its final approval. Limitation of the Study ACKNOWLEDGMENTS This study was not without potential limitations. The authors tried to assess the exit knowledge status of ambulatory patients about dispensed drugs. However, the exit knowledge status was highly subjective to the information retention and recall ability of each patient. This subjective nature of the data could result into an underestimation of the knowledge measurement. This measurement, therefore, considered only those patients who received less than or equal to three drugs from the outpatient pharmacy to assess the exit knowledge of the ambulatory patients since drug information retention ability of the patients was Authors thank data collectors and staff of FHPH, without them this research would not be realized. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpubh. 2018.00254/full#supplementary-material REFERENCES 4. 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Community pharmacist and technician communication with Spanish speaking patients: needs assessment. Am Pharm Assoc. (2012) 52:363–6. doi: 10.1331/JAPhA.2012.10153 Frontiers in Public Health | www.frontiersin.org 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. Copyright © 2018 Hirko, Edessa and Sisay. 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. 8 September 2018 | Volume 6 | Article 254 

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Available online http://arthritis-research.com/content/8/4/R104 Research article Open Access Vol 8 No 4 NOS2 polymorphisms associated with the susceptibility to pulmonary arterial hypertension with systemic sclerosis: contribution to the transcriptional activity Yasushi Kawaguchi1, Akiko Tochimoto1, Masako Hara1, Manabu Kawamoto1, Tomoko Sugiura1, Yasuhiro Katsumata1, Jun Okada2, Hirobumi Kondo2, Mitsuo Okubo3 and Naoyuki Kamatani1 1Institute of Rheumatology, Tokyo Women's Medical University, Tokyo, Japan of Internal Medicine, Kitasato University School of Medicine, Sagamihara, Japan 3Transfusion Medicine and Cell Therapy, Saitama Medical School, Kawagoe, Japan 2Department Corresponding author: Yasushi Kawaguchi, y-kawa@ior.twmu.ac.jp Received: 22 Feb 2006 Revisions requested: 27 Apr 2006 Revisions received: 14 May 2006 Accepted: 2 Jun 2006 Published: 3 Jul 2006 Arthritis Research & Therapy 2006, 8:R104 (doi:10.1186/ar1984) This article is online at: http://arthritis-research.com/content/8/4/R104 © 2006 Kawaguchi 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 Systemic sclerosis (SSc) is a connective tissue disease characterized by tissue fibrosis. One of several complications of SSc, pulmonary arterial hypertension (PAH) can be refractory to treatment, both novel and established. In the present study we investigated the ratio of circulating nitric oxide to endothelin-1 in patients with both SSc and PAH, and determined whether polymorphisms in NOS2 (the nitric oxide synthase 2 gene) are associated with susceptibility to PAH. Endothelin-1 in plasma and nitric oxide metabolites (nitrate and nitrite) in serum were measured. The nitric oxide/endothelin-1 ratio was significantly lower in patients with both SSc and PAH than in patients with SSc only or in healthy control individuals. We confirmed the presence of two single nucleotide polymorphisms at positions 1,026 and -277 and a pentanucleotide repeat (CCTTT) at -2.5 kilobases. There were significant differences in single nucleotide polymorphisms between patients with SSc who had PAH and those who did not, and between patients with both SSc and PAH and healthy control individuals. The CCTTT repeat was significantly shorter in patients with both SSc and PAH than in patients with SSc only or in healthy control individuals. Transcriptional activity were analyzed using the luciferase reporter assay. The transcriptional activity of NOS2 was much greater in fibroblasts transfected by a vector with a long allele of the CCTTT repeat than in those transfected by a vector with a short allele. Polymorphisms in the NOS2 gene are associated with transcriptional activity of the NOS2 gene and with susceptibility to SSc-related PAH. Introduction mia, and pulmonary hypertension due to chronic thrombotic or embolic disease [4]. A major part of pulmonary hypertension as it pertains to SSc corresponds with the pathophysiology of PAH, a disease of the small pulmonary arteries characterized by vascular proliferation, vasoconstriction, remodelling of the pulmonary vessel wall and thrombosis in vessels. Systemic sclerosis (SSc) is an autoimmune disease of unknown aetiology that is characterized by extensive fibrosis of skin and visceral organs, and dysfunction of vascular tone [1]. In its more severe forms, cardiac involvement and respiratory involvement are the most significant determinants of outcome [2]. In particular, pulmonary hypertension is a fatal complication in both diffuse and limited cutaneous SSc [3]. Pulmonary hypertension is generally divided into four major categories: pulmonary arterial hypertension (PAH), pulmonary hypertension associated with left-sided heart disease, pulmonary hypertension associated with lung disease or hypoxae- Vasodilators such as nitric oxide (NO) and prostacyclin, along with prolonged overexpression of vasoconstrictors such as endothelin (ET)-1, not only affect vascular tone but also promote vascular remodelling, both of which have been implicated in the pathogenesis of PAH [5-12]. Previous studies bp = base pairs; DMEM = Dulbecco's modified Eagle's medium; ET = endothelin; FBS = foetal bovine serum; NO = nitric oxide; NOS = nitric oxide synthase; PAH = pulmonary arterial hypertension; PCR = polymerase chain reaction; PPH = primary pulmonary hypertension; SNP = single nucleotide polymorphism; SSc = systemic sclerosis. Page 1 of 10 (page number not for citation purposes) Arthritis Research & Therapy Vol 8 No 4 Kawaguchi et al. identified high levels of ET-1 in the plasma of patients with SSc, especially in those with SSc complicated by PAH [13,14]. However, reported levels of circulating NO in patients with SSc are inconsistent, with several studies [15-18] finding increased levels of NO in patients with SSc and others [19,20] finding low levels, similar to those in healthy individuals. In our previous study [21] NO levels were markedly elevated in patients with early-stage diffuse cutaneous SSc, especially when the SSc was accompanied by active alveolitis, but concentrations of NO in serum were low in late-stage limited cutaneous SSc. No patients suffered the complication of PAH in that study. Characteristic levels of NO and NO/ET1 ratio in patients with both SSc and PAH remain to be established. NO is an endothelial-derived relaxing factor that is synthesized from L-arginine by nitric oxide synthase (NOS) [22]. Three isoforms of NOS have been identified [23]: NOS-1 (neuronal NOS), NOS-2 (inducible NOS) and NOS-3 (endothelial NOS). NOS-2 is the major source of NO production in conditions involving exposure to cytokines; this is because it is induced by a variety of cell types, including the proinflammatory cytokines interleukin-1, tumour necrosis factor-α, interferon-γ, and ET-1 [24]. Two randomized, double-blind, placebo-controlled trials [25,26] evaluated the efficacy of the ET receptor antagonist bosentan in patients with PAH that was either primary or associated with SSc. Another therapeutic strategy in PAH is to increase the activity of endogenous NO, which enhances NOdependent cGMP-mediated pulmonary vasodilatation through inhibition of the breakdown of cGMP by phosphodiesterase type 5 [27]. Although long-term inhaled NO therapy has shown only a small benefit in patients with PAH [28], phosphodiesterase type 5 inhibitors (for example, sildenafil) have been found to improve pulmonary artery pressure in patients with PAH [29]. Because these novel therapies were developed to prolong survival and improve patients' quality of life, we speculate that an imbalance between ET-1 and NO is key to the pathogenesis of SSc complicated by PAH. Polymorphisms in the NOS2 gene promoter are thought to regulate its transcription activity, which is reportedly associated with susceptibility to type 1 diabetes [30] and atopy [31] and with protection against malaria [32]. In the present study we determined the levels of ET-1 and NO in blood from patients with SSc with or without PAH, and we investigated the association between gene polymorphisms in NOS2 and susceptibility to PAH. Materials and methods Study patients Twenty patients with SSc complicated by PAH were recruited. All had been admitted to Aoyama Hospital of Tokyo Women's Medical University or Kitasato University Hospital. As a dis- Page 2 of 10 (page number not for citation purposes) ease control group, 58 patients with SSc but not PAH were selected from patients admitted to Aoyama Hospital. Detailed clinical characteristics of all patients are shown in Table 1. All patients with SSc were of Japanese origin, met the criteria established by the American College of Rheumatology for SSc [33], and were classified as having either diffuse or limited cutaneous SSc according to the classification proposed by LeRoy and coworkers [34]. Ninety-five DNA samples were obtained from healthy volunteers who were unrelated individuals of Japanese origin. All DNA samples were collected, with approvals granted by the appropriate ethical committees of Tokyo Women's Medical University, Kitasato University School of Medicine, and Saitama Medical School. We identified the presence of a complication of PAH in the following manner. All patients with SSc were first evaluated by Doppler echocardiography, and then cardiac catheterization was performed when right ventricular systolic pressure was greater than 30 mmHg, based on Doppler echocardiography. PAH was diagnosed in patients with SSc who satisfied the modified US National Institutes of Health criteria for PAH after cardiac catheterization [35], specifically mean pulmonary artery pressure above 25 mmHg at rest or 30 mmHg after exercise, with normal pulmonary artery wedge pressure. The complication of pulmonary fibrosis was identified using highresolution computed tomography of the chest. Patients with the following complications were excluded: severe pulmonary fibrosis, with functional vital capacity below 70%; left-sided heart disease; chronic thrombotic or embolic disease; renal failure, including a history of scleroderma renal crisis; hypertension; and diabetes. Measurement of plasma endothelin-1 and serum nitric oxide levels Blood samples were obtained from 16 patients with both SSc and PAH and from 26 patients with SSc without PAH who were randomly selected from among patients with SSc who donated DNA samples at the time of admission to Aoyama Hospital with informed consent. No specific diet was given to patients while they were hospitalized. Twenty healthy volunteers (normal control individuals) who had no history of dieting or smoking gave informed consent to participate in the study and gave blood samples. ET-1 levels were measured in plasma using an enzyme-linked immunosorbent assay kit (R&D Systems, Cambridge, MA, USA). Because serum NO is quickly degraded into nitrite and nitrate, we measured the total levels of these NO metabolites as indicators of NO level, using a calorimetric assay kit (Cayman Chemical, Ann Arbor, MI, USA). Sequencing the NOS2 promoter region Genomic DNA was extracted from the blood sample using a DNA extraction kit (Qiagen, Valencia, CA, USA). For direct sequencing, PCR was performed to amplify the promoter region of the NOS2 gene from -100 to -1,335 bp. The forward Available online http://arthritis-research.com/content/8/4/R104 Table 1 Clinical characteristics of patients Characteristic SSc patients Healthy controls With PAH Without PAH 20 (1:19) 58 (5:53) 95 (8:87) 48.6 (17–80) 47.7 (19–77) 40.9 (22–78) 6:14 33:25 42.0 (6–130) 41.9 (5–120) 6 (30) 31 (53) 11 (55) 17 (29) 0 Anti-topoisomerase I antibody 1 (5) 13 (22) 0 Anti-centromere antibody 5 (25) 12 (21) 0 Number at entry (n (male:female) Age (years; mean (range) Diffuse SSc:limited SSc (n) Disease duration (months; mean (range) Pulmonary fibrosis (n (%) Frequency of ANA (n (%) Anti-U1-snRNP antibody ANA, antinuclear antibody; PAH, pulmonary arterial hypertension; SSc, systemic sclerosis. Table 2 Distribution of single nucleotide polymorphisms in NOS2 gene promoter region Subjects -1,026 bp -277 bp Haplotype G/G G/T T/T A/A A/G G/G GA TA GG TG SSc with PAH (n = 20) 20 (100) 0 0 20 (100) 0 0 40 0 0 0 SSc without PAH (n = 58) 43 (74) 11 (19) 4 (7) 43 (74) 10 (17) 5 (9) 96 0 1 19 Healthy controls (n = 95) 69 (73) 23 (24) 3 (3) 73 (77) 19 (20) 3 (3) 161 0 4 25 Values indicate number (%) of genotype or number of haplotype, which consists of two SNPs at -1,026 and -277. There were significant differences between patients with SSc who did and those who did not have PAH in the distribution of two SNPs at -277 and -1,026 (both P = 0.04, by Fisher's exact test). Comparing the distribution of two SNPs between patients with SSc complicated by PAH and healthy control individuals, there was a significant difference at -1,026 (P = 0.02), but there was no difference at -277 (P = 0.053). With respect to haplotype, the frequency of GA was significantly higher in SSc with PAH than in SSc without PAH and in healthy control individuals (P = 0.001 and P = 0.02, respectively). PAH, pulmonary arterial hypertension; SNP, single nucleotide polymorphism; SSc, systemic sclerosis. and reverse primers were 5'-TCATCCACACATTCACTCAAC-3' and 5'-CCAAAGGGAGTGTCCCCAGCTT-3', respectively. The sequences of the PCR products were analyzed using the ABI Prism 3100 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Haplotype typing in pairs of NOS2 polymorphisms We entered the genotype data into the PENHAPLO computer program, developed by Ito and coworkers [36], to estimate haplotype frequency in the population and to calculate the posterior probability of diplotype distribution for each study subject. This program was designed for haplotype typing using a maximum likelihood estimation method based on the expectation maximization algorithm under the assumption of Hardy-Weinberg equilibrium for the population. Analysis of variable numbers of the CCTTT repeat polymorphism of the NOS2 promoter region Genomic DNA was amplified by PCR with the use of a FAM™labelled sense primer (5'-ACCCCTGGAAGCCTACAACTGCAT-3') and an antisense primer (5'-GCCACTGCACCCTAGCCTGTCTCA-3'). The various alleles were resolved by capillary electrophoresis on an ABI Prism 3100 Genetic Analyzer System (Applied Biosystems). Allele sizes were calculated using the GeneScan Analysis computer program, with a GeneScan™-500 ROX™ size standard (Applied Biosystems) as the internal size standard. Page 3 of 10 (page number not for citation purposes) Arthritis Research & Therapy Vol 8 No 4 Kawaguchi et al. Figure 1 A schematic construct of the promoterless pGL3-basic vector vector. The luciferase vector (pGL3) was inserted with variable numbers of CCTTT repeats and the 1.5 kb minimal human NOS2 promoter region (-1,557 to +58 bp). The promoter region contained either T or G at -1026 bp (referred to as pGL3-T and pGL3-G, respectively). Each vector of pGL3-T and pGL3-G had allele G and allele A at -277 bp, respectively. The 6, 8, 10, 12, and 14 repeats of the pentanucleotide (CCTTT) region were obtained by PCR. The resulting constructs were named pGL3-T6, pGL3-G6, pGL3-T8, pGL3G8, pGL3-T10, pGL3-G10, pGL3-T12, pGL3-G12, pGL3-T14 and pGL3-G14 and contain 6, 8, 10, 12 and 14 repeats, respectively. bp, base pairs; kb, kilobases; SNP, single nucleotide polymorphism; VNTR, variable numbers of tandem repeat. Analysis of transcriptional activity of NOS2 in human fibroblasts The 5' flanking region of the NOS2 gene (-1,557 to +58) was prepared by PCR using a set of primers. The forward primer (5'-GATTCTGACTCTTTCCCTGAG-3') is located -1,557 bp from the transcription start site, and the reverse primer (5'GGAATGAGGCTGAGTTCTCTGCGGC-3') is located +58 bp from the transcription start site. Genomic DNA containing the T/G allele at -1026 bp from the transcription start site of the NOS2 gene was used as a PCR template. The PCR product was inserted into a pGL3-Basic Vector (Promega, Madison, WI, USA) that contained the firefly luciferase reporter element, and all constructs were sequenced using the pGL3 forward and reverse sequencing primers. The pGL3 vectors with T or G at -1,026 bp are referred to as pGL3-T and pGL3G, respectively, as shown in Figure 1. The sequences of inserts of pGL3-T and pGL3-G were confirmed by direct sequencing. Each vector of pGL3-T and pGL3-G had allele G and allele A at -277 bp, respectively. The 6, 8, 10, 12 and 14 repeats of the pentanucleotide (CCTTT) region were obtained by PCR using forward (5'-ACCCCTGGAAGCCTACAACTGCAT-3') and reverse (5'-GCCACTGCACCCTAGCCTGTCTCA-3') primers. The PCR products were cloned into the upstream of the inserted NOS2 gene promoter in pGL3-T and pGL3-G. The resulting constructs were named pGL3-T6, pGL3-G6, pGL3-T8, pGL3-G8, pGL3-T10, pGL3-G10, pGL3-T12, pGL3-G12, pGL3-T14 and pGL3-G14, and contain 6, 8, 10, 12 and 14 repeats, respectively. Page 4 of 10 (page number not for citation purposes) Human fibroblasts from three healthy individuals were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% foetal bovine serum (FBS; Sigma, St. Louis, MO, USA). For transient transfections, fibroblasts were cultured in six-well plates with 3 ml Opti-MEM (Invitrogen) containing 4 µg DNA (pGL3 and phRL-TK vectors) and 12 µl Lipofectamine 2000 (Invitrogen). After 4 hours, 3 ml DMEM with 20% FBS in the presence or absence of recombinant interleukin-1β (10 ng/ml; R&D Systems) was added. The medium was changed after 16 hours to DMEM with 10% FBS in the presence or absence of interleukin-1β (5 ng/mL). After an additional 24 hours of culture, the cells were washed twice using cold phosphate-buffered saline and were harvested. Firefly and Renilla luciferase activities were measured using the Dual-Glo Luciferase Assay System (Promega). Fibroblasts were cotransfected with a constitutively active Renilla luciferase vector (phRL-TK), and firefly luciferase activity was normalized by Renilla luciferase activity. Statistics Circulating ET-1 and NO concentrations are given as mean ± standard deviation, and data were compared using the Student's t test. We assessed the significance of the -277A/G and -1026G/T single nucleotide polymorphisms (SNPs) by the Fisher exact test. The relationship between the NO/ET-1 ratio and summed CCTTT repeat length was analyzed using linear regression analysis. An allelic distribution of the number of CCTTT repeats was compared using the Mann-Whitney U test. P < 0.05 was considered statistically significant. Available online http://arthritis-research.com/content/8/4/R104 Figure 2 Circulating levels of ET-1 and NO NO. (a) Plasma levels of ET-1 were measured in patients with SSc with or without PAH. (b) Serum levels of NO metabolites were measured. (c) The ratios of NO/ET-1 are shown. Data are expressed as mean ± standard deviation. ET, endothelin; NO, nitric oxide; NS, not significant; PAH, pulmonary arterial hypertension; SSc, systemic sclerosis. Results Circulating endothelin-1 and nitric oxide concentrations Plasma ET-1 levels were significantly higher in each SSc group than in healthy control individuals (1.4 ± 0.4 pg/ml), as shown in Figure 2a. Moreover, ET-1 levels in patients with both SSc and PH were significantly higher than in patients with SSc but not PAH (4.1 ± 1.7 versus 2.2 ± 0.8 pg/ml; P < 0.001). In contrast, NO levels in patients with both SSc and PAH (114 ± 28 µmol/l) were similar to those in healthy control individuals (95 ± 30 µmol/l), but NO levels in patients with SSc but not PAH (194 ± 89 µmol/l) were significantly higher than in the other two groups (Figure 2b). The NO/ET-1 ratio was significantly lower in patients with both SSc and PAH (32.6 ± 15.7; n = 16) than in patients with SSc but not PAH (87.8 ± 25.0; n = 26) and healthy control individuals (73.6 ± 35.7; n = 20), as shown in Figure 2c. Determination of single nucleotide polymorphisms in the NOS2 promoter region We genotyped the 78 patients with SSc and the 95 control individuals for the promoter region (-100 to -1335 bp) of the NOS2 gene by direct DNA sequencing. We confirmed the presence of two previously reported SNPs at positions -277 and -1026 (Figure 3). The distribution of genotypes is shown in Table 1. The distribution of genotypes at -1026 and -277 was significantly different between patients with SSc who had Page 5 of 10 (page number not for citation purposes) Arthritis Research & Therapy Vol 8 No 4 Kawaguchi et al. Figure 3 Polymorphisms of the 5' flanking region region of of the the human human NOS2 NOS2 gene. gene TATA sequence begins at position -30 bp from the transcription start site of exon 1. bp, base pairs; SNP, single nucleotide polymorphism. PAH and those who did not have PAH (both P = 0.04, by Fisher's exact test), but there was no difference between patients with SSc who did not have PAH and healthy control individuals in the distribution of genotypes at two SNPs. Between healthy control individuals and patients with both SSc and PAH, there was a significant difference in the distribution of genetypes at -1026 (P = 0.02); in contrast, there was no difference at -277 (P = 0.053). Haplotype typing of the NOS2 promoter region We typed the haplotype of the gene, which consists of two SNPs at positions -1,026 and -277. The two SNPs were found to be in linkage disequilibrium. We identified three haplotypes using genes from patients with SSc and healthy control individuals: GA, GG, and TG (Table 2). The frequency of haplotype GA was significantly higher in patients with both SSc and PAH than in patients with SSc but not PAH and in healthy individuals (P = 0.001 and P = 0.02, respectively), as shown in Table 2. Distribution of variable numbers of tandem repeat in the NOS2 promoter region The 15 alleles found in the present study had 6–21 repeats, and the distribution was significantly different between patients with SSc and PAH and healthy control individuals (P < 0.0001) and between patients with SSc with PAH and those with without PAH (P < 0.0001), as shown in Table 3. In contrast, there was no significant difference in distribution between patients with SSc but not PAH and healthy control individuals. If CCTTT repeat length strongly influences NOS2 transcription, then we would expect there to be a significant correlation between CCTTT repeat length and serum NO levels or NO/ET-1 ratios. We calculated the number of summed CCTTT repeats and then analyzed the correlation between that number and serum NO levels or NO/ET-1 ratios. As shown in Figure 4, there was a significant correlation between summed repeat length and both serum NO levels (r = 0.51, P < 0.01; linear regression analysis) and NO/ET-1 ratios (r = 0.83, P < 0.0001) in all patients with SSc. However, in healthy control individuals we identified no significant correlation (data not shown). Page 6 of 10 (page number not for citation purposes) Effects of NOS2 polymorphisms on transcriptional activity of the gene To determine whether the polymorphisms of -277 SNP and 1,026 SNP and variable numbers of tandem repeat were associated with transcription of the NOS2 gene, we evaluated promoter activities using the series of NOS2 promoter-luciferase constructs (as described under Materials and method, above). As shown in Figure 5, NOS2 was almost transcriptionally silent in fibroblasts without stimuli. In contrast, transcription was induced in fibroblasts transfected with vectors, including promoter regions of the NOS2 gene, under stimulation by interleukin-1β. The relative luciferase activities gradually increased with increasing number of CCTTT repeats in both alleles G and T at -1,026. In the case of the same number of CCTTT repeats, the relative luciferase activity was higher in vectors that included the promoter region with allele T at 1,026 than in vectors that included the promoter region with allele G. These findings indicate that transcriptional activity of the NOS gene that includes G at -1,026 and a small number of tandem repeats was low. Discussion In the present study were found that concentrations of NO metabolites were not increased in patients with both SSc and PAH, although plasma ET-1 levels were markedly elevated. Our previous report [21] indicates that serum levels of NO metabolites were significantly higher in patients with SSc than in healthy control individuals, especially in patients with a diffuse cutaneous type, active fibrosing alveolitis, or a short duration since onset. However, the population considered in that study did not include patients with PAH, which could explain why the present findings are inconsistent with those of the previous report. Although a number of reports have been published concerning concentrations of ET-1 or NO in the circulation of patients with SSc [8,13-21], this report is the first to describe an imbalance in the NO/ET-1 ratio in patients with PAH. Over the past decade abnormalities in NO synthesis have been proposed as being important in the pathogenesis and development of pulmonary hypertension, especially primary pulmonary hypertension (PPH). Initially, immunohistochemical studies showed that pulmonary hypertension was associated Available online http://arthritis-research.com/content/8/4/R104 Figure 4 Relationship between NO levels or NO/ET-1 ratios and summed CCTTT repeat length length. The association study between summed lengths of the CCTTT repeat and (a) serum NO levels and (b) NO/ET-1 ratios was performed in 16 patients with SSc with PAH (open circles) and 26 patients with SSc without PAH (black triangles). The results of linear regression analysis of the data are represented by the solid line. Serum NO levels and NO/ET-1 ratios were significantly correlated with summed CCTTT repeat lengths (r = 0.51, P < 0.01; and r = 0.83, P < 0.0001, respectively). ET, endothelin; NO, nitric oxide; PAH, pulmonary arterial hypertension; SSc, systemic sclerosis. Figure 5 Luciferase reporter assay of the NOS2 promoter with gene polymorphisms polymorphisms. The (a) pGL3-T vectors and (b) pGL3-G vectors were cloned by the NOS2 promoter region, including alleles T and G of the single nucleotide polymorphism at -1026 bp. In the series of pGL3-T or pGL3-G vectors, the 6, 8, 10, 12 and 14 repeats of the CCTTT region were cloned upstream of the inserted NOS2 promoter in pGL3-T or pGL3-G. bp, base pairs; IL, interleukin. with diminished expression of NOS-3 [37]. However, other studies found increase in expression of NOS-3 in patients with pulmonary hypertension and in animal models of pulmonary hypertension [38,39]. Despite these contradictory findings, it has been reported that NO levels in blood and the lungs were precisely decreased in patients with PPH and collagen disease related PAH [8-12]. Furthermore, it was determined that NOS-dependent endogenous NO synthesis was decreased in patients with PPH, which suggests that NOS activity may be diminished in patients with PPH [40]. Lung inflammation leadPage 7 of 10 (page number not for citation purposes) Arthritis Research & Therapy Vol 8 No 4 Kawaguchi et al. Table 3 Allele frequencies for the CCTTT-repeat polymorphism Allele SSc patients Healthy controls With PAH Without PAH 6 2 0 0 7 2 0 0 8 8 0 4 9 6 4 8 10 10 11 19 11 5 16 25 12 6 29 42 13 0 22 32 14 1 14 29 15 0 7 13 16 0 4 13 17 0 4 4 18 0 2 1 19 0 2 0 20 0 0 0 21 0 1 0 P < 0.0001 for patients with both SSc and PAH compared with healthy control subjects by the Mann-Whitney U test. P < 0.0001 for patients with both SSc and PAH compared with patients with SSc but not PAH by the Mann-Whitney U test. PAH, pulmonary arterial hypertension; SSc, systemic sclerosis. ing to increased levels of cytokines and oxidants may contribute to the development of both PPH and SSc-related PAH [41]. In the presence of increased levels of inflammatory mediators, NOS activity may be dependent on production of NOS2, which is distinct from NOS-3 (the endothelial form of NOS) because NOS-2 is inducible by inflammatory mediators, and induced levels are much greater than levels of constitutive NOS-3 production. Peripheral mononuclear cells and lesional fibroblasts are capable of aberrant production of inflammatory cytokines in patients with SSc [42-44]. These cytokines may be involved not only in ET-1 synthesis by endothelial cells and fibroblasts but also in induction of NOS-2. Also, excessive production of ET-1 can mediate NOS-2 production through ET receptor B [45]. Although evidence based on those biological properties may promote speculation that levels of ET-1 correlate with levels of NO in the circulation, NO metabolite levels were within normal range in patients with both SSc and PAH patients whose serum contained much ET-1. We hypothesize that this discrepancy may be explained by reduced NOS-2 production resulting from polymorphisms in the NOS2 gene. As a result of sequencing the promoter region of the NOS2 gene from -100 to -1,335, we were able to confirm the pres- Page 8 of 10 (page number not for citation purposes) ence of two SNPs, consistent with previous reports [46]. In the present study, allele A at -277 SNP, allele G at -1,026 SNP and shorter forms of the CCTTT repeat were associated with susceptibility to PAH combined with SSc. The number of CCTTT repeats was previously reported to influence transcription of the NOS2 gene [47]. However, studies of variable numbers of tandem repeat both in vitro and in vivo have yielded conflicting results [48]. To confirm whether those polymorphisms affect transcription of the NOS2 gene in fibroblasts, we constructed a series of luciferase reporter vectors cloned by various numbers of CCTTT combined with the promoter region of the NOS2 gene from +58 to -1,557, which included two kinds of haplotype. Transcriptional activity was lowest in the NOS2 gene containing the six repeats of CCTTT and haplotype GA, which suggests that transcription of the NOS2 gene might be little induced by interleukin-1β in patients with SSc-related PAH. Irrespective of whether patients with SSc had PAH, CCTTT repeat length was well correlated with NO/ET-1 ratio. With regard to the relationship between CCTTT repeat length and serum NO levels, we found no significant difference among SSc patients without PAH, although there were significant differences among all SSc patients and among patients with both SSc and PAH (data not shown). In the setting of aberrant production of ET-1 or cytokines, NO synthesis via NOS-2 induction may be dependent on NOS2 gene polymorphisms. In healthy control individuals, who had no vascular damage, inflammation, or autoimmune disorders, there was no association between CCTTT repeat length and either serum NO levels or NO/ET-1 ratios (data not shown). Because NOS-2 induction is well controlled by ET-1 and cytokines, distinct from NOS-3, which is constitutively produced, it has been suggested that the CCTTT repeat length is more significantly correlated with NO/ET-1 ratios than with serum NO levels. Our observations support the concept that the NOS2 gene polymorphism is a crucial factor in NO synthesis under conditions of vascular damage and chronic inflammation, as well as PAH. It is not possible to determine whether SSc patients without PAH will suffer this complication in the future, and this is a limitation of the present study. The patients enrolled in the study are from a prospective cohort at our institution, and they have been observed for clinical complications, including PAH, in the follow-up clinic. None of the 58 patients with SSc but not PAH has yet been diagnosed with PAH (mean duration of observation: 45 months). Conclusion NO is a key factor in generating PAH complicated by SSc, and the decrease in NO synthesis might be attributable to reduced NOS-2 production, which is dependent on NOS2 gene polymorphisms. Therapeutic options for PAH occurring as a complication of SSc are limited; however, it is not usually the first Available online http://arthritis-research.com/content/8/4/R104 complication, and it develops several years after SSc is diagnosed. We believe that the development of means to predict the occurrence of PAH related to SSc, and hence prevent this complication, would be a great step forward. Although prospective, longitudinal studies are needed, we propose that patients with SSc who exhibit an imbalance between NO and ET-1 production and who have a short length of CCTTT repeat of the NOS2 gene can be treated with a phosphodiesterase type 5 inhibitor before the occurrence of PAH. Competing interests 11. 12. 13. 14. The authors declare that they have no competing interests. Authors' contributions YK designed the study, recruited the patients and drafted the manuscript. AT was responsible for the recruitment and classification of the patients, and determined genotypes of NOS2. MH participated in coordination of the study. MK determined the phenotype of polymorphisms. TS and YK participated in coordination of the study. JO, HK and MO were responsible for the recruitment and classification of patients and healthy volunteers. NK participated in the design and coordination of the study. All authors read and approved the final manuscript. 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pharmaceuticals Article Using a Caenorhabditis elegans Parkinson’s Disease Model to Assess Disease Progression and Therapy Efficiency Samantha Hughes 1,2, *,† , Maritza van Dop 1 , Nikki Kolsters 1 , David van de Klashorst 1 , Anastasia Pogosova 1 and Anouk M. Rijs 3, *,† 1 2 3 * † Citation: Hughes, S.; van Dop, M.; Kolsters, N.; van de Klashorst, D.; Pogosova, A.; Rijs, A.M. Using a Caenorhabditis elegans Parkinson’s Disease Model to Assess Disease Progression and Therapy Efficiency. Pharmaceuticals 2022, 15, 512. HAN BioCentre, HAN University of Applied Sciences, Laan van Scheut 2, 6525 EM Nijmegen, The Netherlands; maritza.vandop@han.nl (M.v.D.); nikkikolsters@live.com (N.K.); david.vandeklashorst@han.nl (D.v.d.K.); anastasia.s.pogosova@gmail.com (A.P.) A-LIFE Amsterdam Institute for Life and Environment, Section Environmental Health and Toxicology, Vrije Univeristeit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands Division of BioAnalytical Chemistry, AIMMS Amsterdam Institute of Molecular and Life Sciences, Vrije Univeristeit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands Correspondence: s.hughes@vu.nl (S.H.); a.m.rijs@vu.nl (A.M.R.) These authors contributed equally to this work. Abstract: Despite Parkinson’s Disease (PD) being the second most common neurodegenerative disease, treatment options are limited. Consequently, there is an urgent need to identify and screen new therapeutic compounds that slow or reverse the pathology of PD. Unfortunately, few new therapeutics are being produced, partly due to the low throughput and/or poor predictability of the currently used model organisms and in vivo screening methods. Our objective was to develop a simple and affordable platform for drug screening utilizing the nematode Caenorhabditis elegans. The effect of Levodopa, the “Gold standard” of PD treatment, was explored in nematodes expressing the disease-causing α-synuclein protein. We focused on two key hallmarks of PD: plaque formation and mobility. Exposure to Levodopa ameliorated the mobility defect in C. elegans, similar to people living with PD who take the drug. Further, long-term Levodopa exposure was not detrimental to lifespan. This C. elegans-based method was used to screen a selection of small-molecule drugs for an impact on α-synuclein aggregation and mobility, identifying several promising compounds worthy of further investigation, most notably Ambroxol. The simple methodology means it can be adopted in many labs to pre-screen candidate compounds for a positive impact on disease progression. https://doi.org/10.3390/ph15050512 Academic Editor: Antoni Camins Espuny Keywords: C. elegans; α-synuclein aggregation; lifespan; mobility; screening platform; therapeutics; Ambroxol; Levodopa Received: 20 March 2022 Accepted: 21 April 2022 Published: 22 April 2022 1. Introduction Publisher’s Note: MDPI stays neutral Parkinson’s Disease (PD) is the second-most prevalent and fastest growing neurodegenerative disease, with 12 million people worldwide expected to be living with PD by 2040 [1,2]. PD is characterized by the accumulation of α-synuclein in the Lewy Bodies, which results in the loss of dopaminergic neurons in the brain [3]. In its pathogenic form, α-synuclein aggregates into the amyloid fibrils, which cause toxicity. Aggregation of α-synuclein is a stochastic event that increases with age, but the rate of aggregation can be accelerated by many additional factors, including the concentration and type of metal ions [4,5], a low pH [6,7], and interactions with lipids [8,9]. Unfortunately, treatment options for people living with PD are very limited, with the most effective therapeutic agent being Levodopa [10–14]. Levodopa acts to replenish dopamine and thus to reduce and delay disease symptoms [15,16], and so it is used in managing symptoms. Targeting the accumulation and aggregation of α-synuclein with a view to slow down, or halt, disease progression is a key goal of pharmacological research [17,18]. with regard to jurisdictional claims in published maps and institutional affiliations. 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/). Pharmaceuticals 2022, 15, 512. https://doi.org/10.3390/ph15050512 https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2022, 15, 512 2 of 19 To identify new therapeutic compounds that slow disease progression and treat the symptoms, it is preferable to screen for candidates in a high-throughput and accurate manner. The nematode Caenorhabditis elegans is ideally suited to such whole-organism screens as it combines the simplicity of cell culture with the complexity of a multi-cellular organism, without the ethical considerations applied to vertebrate animals. C. elegans has a short lifecycle and reproduction time, and a high degree of similarity to humans at the genetic [19,20] and cellular [21] levels. The simple anatomy of C. elegans consists of tissues and organs also found in humans, including a pharynx, intestine, cuticle, and nervous system. With a completely mapped neural network and connectome [22], the nematode worm is frequently used in neurobiology studies. Hallmarks of neuropathologies have been identified in the nematode [23–25], including those for Parkinson’s Disease [26–30], where there are nematodes that recapitulate the most frequent genetic causes of PD, including LRRK2, PARK2, and DJ-1 (reviewed in Maulik et al. [31] and Cooper and van Raamsdonk [32]), as well as modeling α-synuclein expression [27,33,34]. The nematode plays an important role in the drug discovery pipeline, testing the toxicity and efficacy of new pharmaceuticals [35], and has been used to identify inhibitors of α-synuclein aggregation [27,36–39]. Behavioral phenotypes associated with PD can also be observed in nematodes and used as a readout to gain insight into disease pathology, as well as to identify small-molecule compounds that modify disease. In both cases, automation and machine learning aid efficient high-throughput screening [40,41]; however, this requires specialized knowledge or equipment. Many studies have observed changes in the number, size, or intensity of the α-synuclein aggregations following compound exposure as a readout of potential disease modifiers [39,42–45], and each method has its advantages and disadvantages. Measuring the fluorescent intensity of the aggregations is prone to artifacts, while on the other hand, arbitrary choices must be made when counting the number of α-synuclein foci [46], and many use techniques that are not simple or require additional training. To overcome these hurdles, we propose an economical and straightforward whole-system approach to screen drugs with the potential to treat PD, using a standard microscope and freely available software with C. elegans. By utilizing the nematode model for PD that over-expresses the human disease-causing α-synuclein fused to a fluorescent reporter in the body wall muscles, strain NL5901 [27], it is possible to directly monitor α-synuclein aggregation throughout life. This nematode strain mimics the α-synuclein aggregation found in Lewy bodies and idiopathic PD [3,47]; however, it should be noted that this strain does not recapitulate the progressive loss of dopaminergic neurons [48], which is beyond the scope of this work. The open-source ImageJ software [49] was employed to quantify changes in the aggregation of the α-synuclein throughout the lifespan of the nematode in parallel to a behavioral assay as a robust and representative readout of neuromuscular health. Once the parameters were identified, we explored the effect of the most common PD treatment, Levodopa, on the nematodes, to confirm the use of this strain of C. elegans for a drug screening platform. Subsequently, we demonstrated a simple and economically viable platform to screen a variety of small-molecule modulators for PD in an efficient manner, where both the symptoms as well as the cause of the disease were monitored. Repositioning drugs has shown promise for PD treatments [50], with Ambroxol being one such example [51,52]. Ambroxol treatment reduces α-synuclein levels in vitro and in vivo [53–56] and has proceeded to the clinical trial phase [57,58]. To test the applicability of the C. elegans platform, small-molecule modulators used in the treatment of Alzheimer’s and Huntington’s Disease were tested. We confirmed that several of these modulators were able to reduce the α-synuclein aggregation, including Ambroxol, and recover the movement defect in the worm model of PD, demonstrating this economical and simple methodology to screen candidate compounds for a positive impact on disease progression. Pharmaceuticals 2022, 15, x FOR PEER REVIEW Pharmaceuticals 2022, 15, 512 3 of 19 3 of 19 2. Results 2. Results 2.1. Synuclein Plaques Accumulate as Worms Age, Affecting Mobility But Not Overall Lifespan 2.1. Synuclein Plaques Accumulate as Worms Age, Affecting Mobility but Not Overall Lifespan The constitutive expression of α-synuclein fused to a yellow fluorescent protein The constitutive expression of α-synuclein to a yellowoffluorescent protein (strain (strain NL5901) enables visualization of thefused accumulation α-synuclein aggregates NL5901) enables visualization of the accumulation of α-synuclein aggregates throughout throughout the lifespan of the worm [27]. While we were able to confirm the presence of the lifespan in of the While weretoable to on confirm the presence of aggregates in aggregates the worm whole[27]. animal, wewe chose focus the changes in α-synuclein aggrethe whole animal, we chose to focus on the changes in α-synuclein aggregates in the most gates in the most anterior region, the head. The head was chosen as this is the region that anterior the head. The head was chosen as this thecomparison region thatacross couldages. be most could beregion, most easily visualized throughout life and usedisfor The easily visualized throughout life and used for comparison across ages. The differences in differences in α-synuclein aggregation were observed at three key life stages: L4, 1 week α-synuclein aggregation were observed at three key life stages: L4, 1 week (L4 + 7 days), (L4 + 7 days), and 2 weeks (L4 + 14 days) of age. and 2Using weeksconfocal (L4 + 14microscopy, days) of age.Z-stacks were taken through the head of each animal and Using confocal microscopy, Z-stacks were takenusing through the software. head of each animal and the resulting maximal projections were processed ImageJ Representative the resulting maximal projections were processed using ImageJ software. Representative images are shown in Figure 1a of L4, 1-week-old, and 2-week-old nematodes. Worms at images are shown in Figure 1A of L4, 1-week-old, and 2-week-old nematodes. Worms the L4 stage had a large number of small fluorescent foci, the α-synuclein aggregates. at the L4 stage had a large number of small fluorescent foci, the α-synuclein aggregates. Quantification of these foci over a population of worms indicate that L4 worms have an Quantification of these foci over a population of worms indicate that L4 worms have an average of 157 aggregates that are 0.46 µm2 in size (Figures 1B,C). There was a large range average of 157 aggregates that are 0.46 µm2 in size (Figure 1B,C). There was a large range in the number of plaques present at the L4 stage, confirming observations by others in the number of plaques present at the L4 stage, confirming observations by others [27,59]. [27,59]. Moreover, the L4 worms displayed background fluorescence, which together sugMoreover, the L4 worms displayed background fluorescence, which together suggests that gests that the L4 stage is not the ideal stage to probe α-synuclein aggregation. However, the L4 stage is not the ideal stage to probe α-synuclein aggregation. However, including including the L4 stage and applying deconvolution to the image could provide insights the L4 stage and applying deconvolution to the image could provide insights into the full into the full aggregation process, but this is beyond the aim and scope of this work. At 1 aggregation process, but this is beyond the aim and scope of this work. At 1 week of age week of age (L4 + 7 days), worms display an increase in the number and a significant (L4 + 7 days), worms display an increase in the2number and a significant increase in the increase in the size of aggregates, from 0.46 µm to 1.89 µm2. In 2-week-old (L4 + 14 days) size of aggregates, from 0.46 µm2 to 1.89 µm2 . In 2-week-old (L4 + 14 days) nematodes, the nematodes, average size of the α-synuclein aggregates 2.03number µm2, but number is of average size the of the α-synuclein aggregates is 2.03 µm2 , butisthe of the aggregates aggregates is significantly decreased compared to earlier life stages. Our observations are significantly decreased compared to earlier life stages. Our observations are in line with in line with the the processis of aggregation is dynamic, whereby the the hypothesis thathypothesis the processthat of aggregation dynamic, whereby the smaller aggregates smaller aggregates formed during the aging, i.e., from L4 to L4 + 7 days, can act as “seeds” formed during the aging, i.e., from L4 to L4 + 7 days, can act as “seeds” from which the from whichgrow the aggregates grow and fuse together, forming theaggregates observed large aggregates aggregates and fuse together, forming the observed large [60,61]. The full [60,61]. The full range, and especially the smaller species, can be an interesting target for range, and especially the smaller species, can be an interesting target for disease-modifying disease-modifying therapies [62,63]. As the accumulation of the aggregates is visible, this therapies [62,63]. As the accumulation of the aggregates is visible, this provides a useful provides a useful tool to assessofthe effectiveness tool to assess the effectiveness potential drugs. of potential drugs. Figure 1. The α-synuclein aggregates display dynamic changes in size and number across the lifespan of C. elegans. (A) Representative images of the α-synuclein aggregates. Images are maximal projections Pharmaceuticals 2022, 15, x FOR PEER REVIEW Pharmaceuticals 2022, 15, 512 4 of 19 4 of 19 Figure 1. The α-synuclein aggregates display dynamic changes in size and number across the lifespan of C. elegans. (A) Representative images of the α-synuclein aggregates. Images are maximal projections from series of 1 µm Z-stacks taken using a Zeiss LSM 700 fromatworms L4 (i), from a series of 1a µm Z-stacks taken using a Zeiss Axio LSMAxio 700 from worms L4 (i), at 1 week, 1 week, L4 + 7 days (ii), and 2 weeks, L4 + 14 days (iii) of age. Images are of independent animals L4 + 7 days (ii), and 2 weeks, L4 + 14 days (iii) of age. Images are of independent animals and and representative of an n > 14 over a series of 3 independent experiments. The worm strain is representative of an n ≥ 14 over a series of 3 independent experiments. The worm strain is NL5901 NL5901 where α-synuclein is fused to a fluorescent reporter in the body wall muscle. Scale bar, 50 where α-synuclein is fused to a fluorescent reporter in the body wall muscle. Scale bar, 50 µm. µm. (B) Quantification of the number of aggregates and (C) size of aggregates. Maximal projections (B) Quantification the number of aggregates and (C) the sizenumber of aggregates. of of the aggreof confocal imagesofwere quantified using ImageJ with and sizeMaximal (in µm2)projections 2 ) of the aggregates confocal images were quantified using ImageJ with the number and size (in µm gates assessed. The dark blue bars show the L4 stage (n = 15), the blue bars show 1-week-old animals assessed. Then dark show thebars L4 stage = 15), theanimals blue bars 1-week-old (L4 + 7 days; = 18),blue and bars the light blue show (n 2-week-old (L4show + 14 days; n = 14).animals Graphs (L4 + 7 days; n = 18), with and the blue bars 2-week-old animals + 14 days; n =experiments. 14). Graphs shown are averages thelight standard errorshow of the mean (s.e.m.) from(L4 3 independent Statistical 2-sample where the averages are compared to L4-stage shown are analysis averagesused withthe the2-tailed standard error oft-test, the mean (s.e.m.) from 3 independent experiments. worms, unless otherwise indicated. p-value, * p are < 0.05 and *** pto< L4-stage 0.001. Statistical analysis used the 2-tailed Asterisks 2-sample indicate t-test, where the where averages compared worms, unless otherwise indicated. Asterisks indicate p-value, where * p < 0.05 and *** p < 0.001. Sarcopenia, the progressive loss of muscle mass and function [64,65], is prevalent in the progressive of muscle and function is prevalent olderSarcopenia, adults living with PD and isloss associated withmass the severity of the [64,65], disease [66,67]. In C. in older adults living with PD and is associated with the severity of the disease [66,67]. elegans, sarcopenia corresponds to a reduction in mobility, which is assessed by counting In elegans,ofsarcopenia corresponds reduction in A mobility, which is assessed by theC.number body bends, or thrashes,toinaliquid media. bend was counted as a single counting the touch number of body bends, or thrashes, liquid media.Movie A bend was a head-to-tail using an ImageJ plugin (see in Supplemental S1). Tocounted exploreas the single head-to-tail touch using an ImageJ plugin (see Supplemental Movie S1). To explore link between sarcopenia and disease progression, wild-type worms and those that express the between sarcopeniaα-synuclein and diseasewere progression, worms those that the link human disease-causing assessedwild-type for differences inand body bending express the human disease-causing α-synuclein were assessed for differences in body (Figure 2). At the L4 stage, both wild-type and the nematode model for PD display a simbending (Figure 2). At the L4 stage, both wild-type and the nematode model for PD display ilar thrash rate, with an average of 43 body bends per minute (BBPM). As the worms age, a similar thrash rate, with an average of 43 body bends per minute (BBPM). As the worms their mobility declines, which is clearly observed by the reduction in thrash rate of 5% and age, their mobility declines, which is clearly observed by the reduction in thrash rate of 5% 43% after 1 week and 2 weeks, respectively. Interestingly, the worms that express α-synuand 43% after 1 week and 2 weeks, respectively. Interestingly, the worms that express αclein (strain, NL5901) display a more rapid decline in mobility, with an 18% reduction in synuclein (strain, NL5901) display a more rapid decline in mobility, with an 18% reduction body bends at 1 week of age, and a 65% reduction in body bends at 2 weeks of age. in body bends at 1 week of age, and a 65% reduction in body bends at 2 weeks of age. Figure 2. 2. The worm age, with a more pronounced effect in the Figure The number numberof ofbody bodybends bendsreduced reducedwith with worm age, with a more pronounced effect in worm model for Parkinson’s Disease. The number of body bends per minute were were assessed in wildthe worm model for Parkinson’s Disease. The number of body bends per minute assessed in type (red bars; strain, N2) and expressing the human α-synuclein in body wall muscle (blue wild-type (red bars; strain, N2)worms and worms expressing the human α-synuclein in body wall muscle bars; strain, NL5901) animals at L4, at 1 week + 7(L4 days), 2 weeks + 14(L4 days) age. Counts (blue bars; strain, NL5901) animals L4, 1 (L4 week + 7 and days), and 2 (L4 weeks + 14ofdays) of age. were assessed using wRMTrck in ImageJ and averages plotted with the standard error of the mean Counts were assessed using wRMTrck in ImageJ and averages plotted with the standard error of the (s.e.m.). Statistical analysis used the 2-tailed 2-sample t-test where *** indicates a p value of p < 0.001 mean (s.e.m.). Statistical analysis used the 2-tailed 2-sample t-test where *** indicates a p value of when comparing the same strain to L4, or as indicated, and n.s. designates not significant. N ≥ 60 pover < 0.001 when comparing the same strain to L4, or as indicated, and n.s. designates not significant. 3 independent replicates. n ≥ 60 over 3 independent replicates. As the nematode model for PD had an enhanced age-related reduction in mobility, As the nematode model for PD had an enhanced age-related reduction in mobility, similar to people living with the disease, we sought to examine if this was reflected in similar to people living with the disease, we sought to examine if this was reflected in lifespan differences. differences. The forfor thethe wild-type worms as for the lifespan Themaximal maximallifespan lifespanwas wassimilar similar wild-type worms as for Pharmaceuticals 2022, 15, 512 5 of 19 Pharmaceuticals 2022, 15, x FOR PEER REVIEW 5 of 19 the worms with the α-synuclein construct (strain, NL5901), as was the day at which 50% worms with the α-synuclein construct (strain, NL5901), as was the day at which 50% mormortality was observed (Figure 3, Supplementary Table These suggest that the tality was observed (Figure 3, Supplementary Table S1). These dataS1). suggest thatdata the prespresence the α-synuclein aggregates does not impact lifespan. ence of theof α-synuclein aggregates does not impact lifespan. Figure 3.3.C.C.elegans thatthat express the human α-synuclein have no difference in lifespan compared to compared to Figure elegans express the human α-synuclein have no difference in lifespan control worms. Wild-type animals (strain, N2; red line) and those that express the human α-synucontrol worms. Wild-type animals (strain, N2; red line) and those that express the human α-synuclein clein (strain, NL5901; blue line) were observed for survival on NGM in the presence of FUdR, a (strain, NL5901; blue line) werefrom observed for The survival on NGM in the presence of FUdR, compound that prevents offspring hatching. life stages of nematodes are indicated along a compound the top of the graph and the 50% survival point highlighted by a dashed line. Details of the mean the top of the that prevents offspring from hatching. The life stages of nematodes are indicated along and maximal lifespan are shown in Supplementary Table S1. graph and the 50% survival point highlighted by a dashed line. Details of the mean and maximal lifespan are shown Supplementary Table S1. 2.2. An Affordable andinSimple Platform to Assess Disease Progression and Therapy Efficiency To gain a detailed of α-synuclein aggregation, a confocal 2.2. An Affordable andunderstanding Simple Platform to Assess Disease Progression andmicroscope Therapy Efficiency was used. While this provides an accurate assessment of the α-synuclein aggregates, it is To gain a detailed understanding of α-synuclein aggregation, a confocal a time-consuming and labor-intensive process, as well as being relatively expensive to microscope was used.Most While this provides an accurate assessment of the α-synuclein aggregates, it purchase. laboratories have access to a conventional fluorescent microscope, which is time-consuming well asexamined being relatively hasa the added benefit of and beinglabor-intensive relatively simple process, to use. Weas therefore whether expensive similar results could obtained usinghave conventional microscopy, in our case, microscope, to purchase. Mostbelaboratories access fluorescent to a conventional fluorescent a Zeiss has Imager.M2 microscope and Axio LSM700,simple respectively. Images ob- examined which the added benefit ofZeiss being relatively to use. We were therefore tained of L4 and 1-week-old (L4 + 7 days) nematodes on both systems and analyzed with whether similar results could be obtained using conventional fluorescent microscopy, in ImageJ. It was not possible to adequately quantify the α-synuclein aggregation in the imour case, a Zeiss Imager.M2 microscope and Zeiss Axio LSM700, respectively. Images were ages of L4 animals generated with the conventional microscope, due to elevated levels of obtained offluorescence L4 and 1-week-old 7 days) nematodes on both systems and analyzed background obstructing(L4 the+actual aggregates. However, the images of 1with ImageJ. It was not possible to adequately quantify the α-synuclein aggregation in the week-old nematodes generated using the conventional microscope could be quantified, images of L4 generated the conventional microscope, due to elevated levels as they have ananimals increase in the size of with aggregates compared to the L4 stage (Supplemental Figure S1). It is challenging to directly comparethe results, as images from theHowever, conventional of background fluorescence obstructing actual aggregates. the images of microscope can only be obtained fromusing one focal plane, while a microscope confocal takescould images 1-week-old nematodes generated the conventional be quantified, through the whole thickness of the worm. However, our results demonstrate that by using as they have an increase in the size of aggregates compared to the L4 stage (Supplemental a conventional fluorescent microscope as found in most laboratories, it is possible to Figure S1). It is challenging to directly compare results, as images from the conventional screen worms for changes in the α-synuclein aggregation more quickly than on a confocal microscope can be obtained from one focalis plane, while confocal takes images microscope. The useonly of ImageJ to quantify the aggregates also simple and arobust for any through the whole thickness of the worm. However, our results demonstrate that by operator to use. For this reason, all further experiments were undertaken using a convenusing a conventional fluorescent microscope found in most laboratories, it is possible to tional fluorescent microscope, the Zeiss Imager.M2 as microscope. screen worms for changes in the α-synuclein aggregation more quickly than on a confocal microscope. The use of ImageJ to quantify the aggregates is also simple and robust for any operator to use. For this reason, all further experiments were undertaken using a conventional fluorescent microscope, the Zeiss Imager.M2 microscope. To explore the effect of possible small-molecule inhibitors on the aggregation and mobility of C. elegans, we chose to assess worms at 1 week of age (L4 + 7 days). The reason for this was three-fold; first, animals at 1 week of age are best and more reliable to observe and quantify the changes in fluorescently tagged α-synuclein and body bends; secondly, Pharmaceuticals 2022, 15, 512 To explore the effect of possible small-molecule inhibitors on the aggregation and mobility of C. elegans, we chose to assess worms at 1 week of age (L4 + 7 days). The reason for this was three-fold; first, animals at 1 week of age are best and more reliable to observe and quantify the changes in fluorescently tagged α-synuclein and body bends; secondly, 6 of 19 L4 + 7 days showed the best balance of the number of live and dead worms; and thirdly, worms at this stage of development are predicted to be the age of worm that best represents humans with PD [37,68,69]. L4 + 7 days showed the best balance of the number of live and dead worms; and thirdly, worms at this stage of Recovers development are predicted to in be athe of worm that best represents 2.3. Levodopa Exposure the Mobility Defect C.age elegans PD Model, While the αhumans with PD [37,68,69]. Synuclein Aggregates are Not Affected 2.3.To Levodopa Recovers Defect in a C. to elegans PDdrug Model, While the Levodopa test theExposure applicability ofthe ourMobility analysis platform probe effectivity, α-Synuclein Aggregates Are Not Affected was chosen as it is the “Gold standard” treatment for Parkinson’s Disease [70,71]. ConTo test applicability platform to probe drug Levodopa centrations ofthe Levodopa (0.1,of 1, our andanalysis 3 mM) were selected based oneffectivity, the literature [39,72,73] was chosen as it is the “Gold standard” treatment for Parkinson’s Disease [70,71]. Concenand tested for an effect on α-synuclein aggregation and body bending. There was no sigtrations of Levodopa (0.1, 1, and 3 mM) were selected based on the literature [39,72,73] and nificant change in the number nor size of α-synuclein aggregates compared to control tested for an effect on α-synuclein aggregation and body bending. There was no significant conditions 0.1 mM and mMofLevodopa (Figure 4a). However, continuous exposure to change inat the number nor1size α-synuclein aggregates compared to control conditions 3 mM Levodopa from L4 for 1 week (L4 + 7 days) resulted in a small but significant at 0.1 mM and 1 mM Levodopa (Figure 4a). However, continuous exposure to 3 mM (p < 0.05) decrease in aggregate number a corresponding increase their size. Levodopa from L4 for 1 week (L4 + but 7 days) resulted in a small but in significant (p There < 0.05) was nodecrease effect ofinthe drug on the mobility of wild-type C. elegans (Figure 4b), was but,no ineffect contrast, aggregate number but a corresponding increase in their size. There of the drug on the mobility of wild-type C. elegans (Figure 4b), but, in contrast, exposure towith exposure to Levodopa at all concentrations recovered the body bending in the worms Levodopa at all concentrations recovered bending animals in the worms α-synuclein aggregates. The body bendsthe inbody the NL5901 at 1 with weekα-synuclein of age was 25 aggregates. The body bends in the NL5901 animals at 1 week of age was 25 BBPM, BBPM, which was increased to 44 BBPM following exposure to 0.1 and 1 mM which Levodopa increased to 344mM BBPM followingalso exposure to 0.1 1 mM Levodopa forto 1 week, with Toforwas 1 week, with Levodopa causing theand thrashes to increase 37 BBPM. 3 mM Levodopa also causing the thrashes to increase to 37 BBPM. Together, the recovery in gether, the recovery in body bending in the NL5901 nematodes exposed to all concentrabody bending in the NL5901 nematodes exposed to all concentrations of Levodopa was not tions of Levodopa was not significantly different to the body bends of wild-type animals significantly different to the body bends of wild-type animals at the same age (Figure 4b), at thus the same age (Figure 4b), thus demonstrating that the Levodopa able to the modemonstrating that Levodopa is able to recover mobilityisdefect onrecover the worms bility defect on worms expressing α-synuclein. the human disease-causing α-synuclein. expressing thethe human disease-causing Figure 4. The dose–response curve for Levodopa on thrashing and α-synuclein aggregates. (a) Hallmarks of the Parkinson’s Disease shown by the α-synuclein aggregates. Representative Zeiss image Pharmaceuticals 2022, 15, 512 7 of 19 of the head of 1-week-old (L4 + 7 days) animal. Representative images of the NL5901 strain under (i) control conditions where the NGM contains FUdR, (ii) 0.1 mM, (iii) 1 mM, or (iv) 3 mM Levodopa. Scale bar, 50 µm. Quantification of the number and size of α-synuclein aggregates from Zeiss images. In both cases, the average is plotted with the standard error of the mean (s.e.m.) with the FUdR control (blue bars; n = 62) and 1 mM Levodopa (purple bars; n = 24). There is no significant difference in number or size of the aggregates following exposure to 0.1 nor 1 mM Levodopa, but a significant increase in aggregate size (shown by * where p < 0.05) at 3 mM Levodopa. (b) The average body bends per minute (BBPM) were assessed in either N2 (circles) or NL5901 (triangles) nematode strains in control (NGM supplemented with 100 µM FUdR) or Levodopa-supplemented NGM where n ≥ 20 for all conditions. BBPM was assessed using wRMTrck in ImageJ and averages plotted with the standard error of the mean in GraphPad Prism v9. Each point represents a single worm assessed. The only statistical difference is comparing N2 and NL5901 in the absence of drugs, where the thrashing in NL5901 is significantly reduced. Asterisks *** indicate a p value of <0.001. (c) Worms were assayed for survival in the presence of FUdR (wild-type stain, N2, red line; PD model strain NL5901, blue line) and 1 mM Levodopa (wild-type stain, N2, black line; PD model strain NL5901, purple line). C. elegans that express the human α-synuclein do not show a significant difference in lifespan compared to wild-type worms. Details of the mean and maximal lifespan are shown in Supplementary Table S1. To assess the effect of Levodopa on lifespan, both wild-type and NL5901 worms were continually exposed to the drug at 1 mM Levodopa from L4 onwards. This concentration was selected based on the mobility and plaque data, as well as data from the literature [72]. When exposed to Levodopa, wild-type worms have a maximal lifespan of 25 days, while the NL5901 worms (the nematode PD model) have a maximal lifespan of 20 days (Figure 4c, Supplementary Table S1). While this is significantly different, it is likely to be due to a single wild-type animal. It is of interest to note that the age at 50% mortality is greater in NL5901 worms, at 13 days compared to 11 days for the wild-type worms exposed to Levodopa. Our data on Levodopa treatment in C. elegans that express the human disease-causing α-synuclein are in line with the effect of Levodopa in humans living with Parkinson’s Disease [12,74,75]. In this way, we have demonstrated that our platform is suitable to screen compounds for a therapeutic effect. The advantage of our platform is that it can be easily applied in a small laboratory setting to screen compounds for a positive effect on PD. To further test the applicability for this, we chose to screen a selection of compounds that may confer a positive effect on α-synuclein aggregation and body bending. 2.4. Screening of Small-Molecule Modulators for a Positive Effect on the Hallmarks of PD in C. elegans The small-molecule modulators that were selected in this study are used to treat PD and other neurodegenerative diseases. Ambroxol [58,76] and Betulin [44,77] were selected as these are treatments for PD. Valproic acid [78,79], Bexarotene [80,81], and Galantamine [82–84] were included as potential therapies for people living with PD but are more commonly used to treat Alzheimer’s Disease. Lastly, we tested Tetrabenazine, which is predominantly a treatment for Huntington’s Disease [85,86] but has shown promise for PD patients who have Levodopa-induced dyskinesias [87]. In all cases, worms were exposed to compounds for 1 week (L4 + 7 days) before being used for quantification of the α-synuclein aggregation (Figure 5) and analysis of body bending (Table 1 and Supplemental Figure S2). Lifespan was only evaluated for Ambroxol (Supplemental Figure S3). As, for both Levodopa and Ambroxol, lifespan was not altered (shortened or lengthened), we excluded this for the other small-molecule modulators in this study. However, it can be an interesting hallmark to explore for future lead compounds. Pharmaceuticals Pharmaceuticals 2022, 2022,15, 15,512 x FOR PEER REVIEW of 19 88 of 19 Figure Figure 5. 5. A A survey survey of of the the effect effect of of drugs drugson onα-synuclein α-synuclein aggregates. aggregates. Quantification Quantification of the the number number (left panel) panel) and and size size (right (right panel) panel) of of α-synuclein α-synuclein aggregates aggregates from from Zeiss Zeiss images images after after exposure exposure to to (left drugs for for 11 week week (L4 (L4 ++ 77 days) days) for for each each drug. drug. In In all all cases, cases, the the average average is is plotted plotted with with the the standard standard drugs error of the mean (s.e.m.). The statistical tests are a one-way ANOVA where * p < 0.05, ** p < 0.01, *** Pharmaceuticals 2022, 15, 512 9 of 19 error of the mean (s.e.m.). The statistical tests are a one-way ANOVA where * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001. The blue bars show the control, FUdR, n = 62 over 6 independent replicates. All dark grey bars represent exposure to 0.1 mM for 1 week, light grey indicates 1 mM exposure, and white bars are for worms exposed to 3 mM of the drug. (a) Exposure to Ambroxol (n = 20, 20, 12). Using ANOVA, there was no significant difference between the aggregate number of Ambroxol-exposed worms compared to the control. However, a 2-tailed 2-sample t-test gave a p = 0.029 between FUdR and 3 mM Ambroxol-exposed worms; (b) Betulin (n = 33, 36). Note that 3 mM was not tested, as this was found to be toxic to C. elegans by other groups; (c) Bexarotene (n = 20, 22, 21, 15); (d) Galantamine (n = 13, 7, 11); (e) Tetrabenazine (n = 22, 22, 15) showed no significant difference between the FUdR control and Valproic Acid-exposed worms nor did a t-test; (f) Valproic Acid (n = 23, 22, 22). Table 1. Survey of the effect of drugs on body bending. Worms (strain N2 and NL5901) were exposed to each drug for 1 week (L4 + 7 days) after which they were assessed for body bending. All drugs were tested at 0.1, 1, and 3 mM, with the exception of Ambroxol, Betulin, and Tetrabenazine, which were not tested at 3 mM (shown by an “n/a”), as the worms paralyzed immediately upon being placed in the M9 buffer. For graphical representation, see Supplemental Figure S2. All experiments were repeated once, with a minimum of 11 worms per condition. The controls (FUdR and DMSO) were run with each experiment but are combined in the table. N2 (Wild-Type) 0.1 mM Control 0.1% DMSO 1% DMSO 3% DMSO Ambroxol Betulin Bexarotene Galantamine Tetrabenazine Valproic acid 1 mM NL5901 (α-Synuclein) 3 mM 0.1 mM 32 32 38 31 33 40 33 22 36 40 34 38 41 29 39 39 1 mM 3 mM 27 37 37 22 n/a n/a 32 32 n/a 46 23 40 33 21 24 34 21 42 39 17 22 41 n/a n/a 38 20 n/a 40 Exposure to Ambroxol showed a reduction in the α-synuclein aggregates (Figure 5a), which were almost completely ameliorated when worms were exposed to 3 mM of the drug (Supplemental Figure S4). While there was no observable recovery of the body bending at 0.1 and 1 mM (Table 1, Supplemental Figure S2), those worms exposed to 3 mM Ambroxol were paralyzed directly upon being placed in the M9 buffer for the mobility assay. It is possible that the mobility defects are a consequence of the elevated solvent percentage [88,89], which, in this case, is 3% DMSO, due to the limited solubility of Ambroxol in lower DMSO concentrations. Worms exposed to 3% DMSO did not display the same level of paralysis nor such a reduction in the α-synuclein aggregates (Supplemental Figure S5). Similarly, we were unable to test Betulin at 3 mM due to its toxicity [44], but at lower concentrations, there was a significant reduction in the number of aggregates (Figure 5b). It was of significant interest to see that exposure to Betulin also resulted in a recovered mobility of the worms that expressed the α-synuclein to comparable levels in the wild-type worms (Table 1, Supplemental Figure S2). Bexarotene at all concentrations significantly reduced the number of aggregates, although at 3 mM, the present aggregates were also significantly larger (Figure 5c). Accordingly, there was a recovery in the mobility defect of this nematode PD model (Table 1, Supplemental Figure S2). Exposure to Galantamine had a similar effect to Bexarotene, but there was no recovery in the mobility of the animals (Figure 5d, Table 1 and Supplemental Figure S2). Exposure to 0.1 mM of Tetrabenazine had no effect on α-synuclein size nor number (Figure 5e). However, higher Pharmaceuticals 2022, 15, 512 10 of 19 concentrations of Tetrabenazine did result in a significant increase in aggregate size, while their number remained constant (Figure 5e). There was no recovery in the body bending of NL5901 worms when exposed to Tetrabenazine, although the 3 mM concentration could not be tested effectively, as the worms were paralyzed when placed in M9 buffer (Table 1, Supplemental Figure S2). Valproic acid was a very interesting compound in that at all concentrations tested, it caused the aggregates to be smaller in number, but with an increase in size. Coincidently, there was a recovery of the mobility of the animals (Figure 5f, Table 1 and Supplemental Figure S2). Taken together, these data show that the C. elegans platform is a robust method to observe changes in the size and number of α-synuclein aggregates, as well as mobility, with a view implicating lead compounds for further, detailed analysis. 3. Discussion C. elegans are utilized in phenotypic drug screening as they are small, have high genetic homology with humans, and raise no ethical concerns [90]. While C. elegans are used in screens to identify pharmaceuticals for Parkinson’s Disease, many protocols use techniques that require specialist knowledge and/or equipment. We wished to develop an accessible and affordable approach to test the ability of pharmaceuticals to reduce the symptoms and hallmarks of Parkinson’s Disease. A multi-phenotype platform is especially important, as currently no therapeutics exist that are capable of addressing both the symptoms (mobility defects) and cause of the disease (α-synuclein aggregation). This study has provided additional information into the phenotypic effects of the Parkinson’s Disease model C. elegans (strain, NL5901). The accumulation of α-synuclein was assessed during the lifetime of the nematode in an affordable, simple, and robust manner. While previous groups have explored the change in the number of α-synuclein aggregates in aging worms, these investigations tend to focus on a shorter period of time, such as for 4 days post-L4 [27,59], despite younger worms being less representative of human PD in an aging population. We confirm that there is no correlation between the presence of aggregates and maximal lifespan, in line with other studies [59,91]. Further, it should also be noted that the disease pathology in C. elegans also arises from the fact that normal cellular proteins are more prone to aggregation due to the decline in proteostasis mechanisms [92,93]. These findings in C. elegans are parallel to observations in humans whereby the increase in protein aggregation over time corresponds to functional decline [92,94]. To test if this platform assesses potential small-molecule compounds that could interfere with the aggregation process, worms were continuously exposed to the small-molecule drugs from L4 until the drug effect was probed in 1-week-old (L4 + 7 days) worms. Initially, the platform was tested using Levodopa, the “Gold Standard” treatment for PD [70,71]. While Levodopa had no effect on the α-synuclein aggregations, there was a significant recovery of the body bending defect. These results are in line with those from other nematode studies and in mammalian experiments [12,13,39,72] and are analogous to results in humans with PD that take Levodopa [12,74,75]. The strain used here has α-synuclein being expressed in the body wall muscles and, therefore, it raises the question as to how exposure to Levodopa would recover the mobility defect in muscles that are not directly innervated by dopaminergic neurons. In C. elegans, dopamine receptors can be found throughout the nematode nervous system, and a connection has been shown between dopamine, motor circuitry, and behaviors including locomotion [95–97]. Indeed, two key dopamine receptors, D1- and D2-like receptors (DOP-1 and DOP-3, respectively in C. elegans), are present in cholinergic and GABAergic motor neurons, which are key in the control of worm mobility [72,97]. In worms that express α-synuclein, D1 receptor (D1R) expression was increased when worms were exposed to L-DOPA [72], in agreement with findings in mammals [98,99]. Exogeneous dopamine has been shown to control motor function in somatic motor neurons [100], and the depletion of dopamine in people with PD results in the motor symptoms associated with PD. It is therefore possible that the exogeneous Pharmaceuticals 2022, 15, 512 11 of 19 dopamine provided to C. elegans in the form of Levodopa is able to stimulate muscles and result in an increase in the thrashing observed in the worms that express α-synuclein, while wild-type worms that do not have the integrated transgene do not show such an effect. While this is of great interest and worthy of further investigation, it is currently beyond the scope of this work. However, it does show the relevance of the C. elegans model for screening for treatments for PD. There was no detrimental effect of long-term Levodopa exposure on the overall health and lifespan of either wild-type or PD nematodes. Indeed, Levodopa has a marginal positive effect on health span in PD worms, which may relate to an enhanced quality of life in people living with PD. This is of note, as the age of onset of Levodopa treatment may affect the outcome for the patient in terms of quality of life [101] and the duration of treatment may be of importance to prevent Levodopa-induced dyskinesia [102]. These findings demonstrated that our platform is a suitable method by which to test the effectiveness of drugs on both the symptom (mobility) and cause (α-synuclein aggregation) of PD. This will provide a sound basis from which to move target compounds from an initial screen into higher mammalian models to be able to translate the data to humans and ultimately find a cure for PD. Further, this platform may also prove applicable to explore the impact of small-molecule inhibitors on other diseases. For example, the accumulation of α-synuclein contributes to the aggregation of islet amyloid polypeptide (IAPP), which has been linked to type 2 diabetes [103,104]. To test the platform further, a number of drugs were screened for their effectiveness to rescue the body bending deficit in α-synuclein-expressing worms and a reduction in the number and size of the aggregates. One of the compounds, Ambroxol, was extremely interesting, especially as it is a highly promising new treatment for PD [57,58]. In our platform, the α-synuclein aggregates were almost completely abolished in worms exposed to 3 mM Ambroxol (Figure 5a and Supplemental Figure S4). However, when tested in the mobility assay, these animals appeared to be paralyzed. It is possible that the mobility defects are a consequence of the elevated solvent percentage [88,89], which, in this case, is 3% DMSO. This was unavoidable due to the limited solubility of Ambroxol, and alternatives may be more toxic to the nematodes. Additional studies will be required to separate the response of C. elegans to Ambroxol and DMSO. Further, it is of interest to explore the effect of Ambroxol on α-synuclein aggregation, in terms of the aggregates themselves [105], but also to observe how the kinetics of aggregation alters in the presence of Ambroxol [106–108]. From our screen, we also identified other drugs that are of interest as treatments for PD. We showed that Valproic Acid reduces the number of α-synuclein aggregations, suggesting a positive effect at modulating disease progression. This is in line with previous studies demonstrating that Valproic Acid has a neuroprotective effect in nematodes [109] and in rodents [78,110]. The recovery of the mobility defect in worms that express α-synuclein in the presence of Valproic Acid also recapitulates the results observed in rats [79]. Bexarotene, Galantamine, and Tetrabenazine are interesting candidates for further research as these compounds caused a reduction in the number of α-synuclein aggregates but an increase in size, suggestive of a disruption of the aggregation process. This is striking as low doses of Bexarotene have been shown to prevent dopamine neuron degeneration in rats [81]. Further confirmation of Bexarotene as a potential drug for PD is that the mobility defect is rescued in C. elegans expressing α-synuclein, similar to the situation in rats [81]. Unlike Bexarotene, the mobility was not rescued in worms exposed to Galantamine or Tetrabenazine. Galantamine functions as an acetylcholinesterase inhibitor [111], and there is evidence that Galantamine, as with other cholinesterase inhibitors, is able to treat people with Parkinson’s Disease dementia [83,112]. While there are some cholinergic motor neurons in C. elegans, it is likely that the assay used in this platform is not sensitive enough to observe behavioral changes. Therefore, in future studies, more detailed experiments would be needed. An effect on body bending following exposure to Tetrabenazine was expected as this compound increases extracellular dopamine [113]. However, as Tetrabenazine is a VMAT2 agonist, which inhibits dopamine uptake, it is therefore not likely to affect nematode mobility [114]. Taken Pharmaceuticals 2022, 15, 512 12 of 19 together, using C. elegans to explore the effects of compounds on two hallmarks of PD in parallel enables the drug discovery pipeline to be more streamlined and contributes to the fast identification of possible lead compounds for further mammalian studies. 4. Materials and Methods 4.1. Strains and Nematode Preparation Strains used in this study were wild-type N2 var. Bristol and NL5901 (pkIs2386 (unc-54p::alpha-synuclein::YFP + unc-119(+) )). Nematode strains were provided by the Caenorhabditis Genetics Centre (CGC) and maintained on Nematode Growth Media (NGM) agar prepared according to standard protocols [115] and plates seeded with OP50 E. coli as a bacterial food source. To synchronize the worm population, gravid animals were washed from plates and bleached with alkaline hypochlorite solution (4 mL of 5% sodium hypochlorite, 1 mL of 4 M sodium hydroxide, and 5 mL of dH2 O) according to standard protocols [116]. The eggs were left to hatch overnight in M9 buffer in the absence of food at room temperature (19–20 ◦ C). The following day, the synchronized L1 larvae could then be placed directly onto NGM. 4.2. Small-Molecule Modulators The small-molecule drugs studied in this work are summarized in Table 2. They were dissolved without further purification to 100 mM in DMSO or water and subsequently diluted to 0.1, 1, and 3 mM. Betulin required sonication to fully dissolve. Stocks were made fresh for each use and then filter-sterilized before being added to molten NGM to the required concentration. Table 2. Small-molecule inhibitors used in the study. Drug Solvent Order Number Molecular Weight (g/mol) Company Disease Ambroxol Betulin Bexarotene Galantamine Levodopa Tetrabenazine DMSO DMSO DMSO Water Water DMSO A9797 B9757 SML0282 Y0001279 PHR1271 T2952 378.10 442.72 348.48 368.27 197.19 317.42 Sigma Aldrich Sigma Aldrich Sigma Aldrich Sigma Aldrich Merck Sigma Aldrich Valproic Acid Water PHR1061 144.21 Sigma Aldrich Parkinson’s Parkinson’s Alzheimer’s Alzheimer’s Parkinson’s Huntington’s Alzheimer’s Parkinson’s 4.3. Treatment Age-synchronized L1s were cultivated on standard NGM plates. When at the L4 stage, the worms were transferred to seeded NGM plates containing the drug of interest at the desired concentration with 100 µM 20 -Deoxy-5-fluorouridine (FUdR) to prevent progeny from hatching or NGM supplemented with FUdR and compounds of interest. In all cases, NGM supplemented with FUdR were considered the control plates. Plates were incubated at 20 ◦ C until the worms were scarified for microscopy at 1 week (L4 + 7 days) or 2 weeks (L4 + 14 days) of age, as described in each experiment. 4.4. Thrashing Assay and Analysis L4 worms were transferred to drug-supplemented NGM and incubated at 20 ◦ C until the desired stage was reached. The number of body bends was quantified by placing the worms into M9 buffer (40 µL) on an unseeded NGM plate at room temperature. Videos were taken using a Leica S8aP0 binocular microscope with a Leica DMC2900 camera and the LAS v4.12 software [117]. The 90 s movies were analyzed using ImageJ v1.53 software with the wRMTrck plugin (build 110622) [49] used to quantify the number of body bends. Please see Supplemental Movie S1 to observe how body bends are counted by the plugin. Pharmaceuticals 2022, 15, 512 13 of 19 The frames were normalized to that of the highest light intensity and the background was removed. Movies were set to 28 frames per second with a bend threshold of 1. Each worm was assessed for number of body bends per minute (BBPM). For the drug assessment, both N2 (wild-type) and NL5901 (α-synuclein-expressing worms) were assessed after 1 week of exposure (L4 + 7 days) with at least 10 worms per condition. Any worms with less than 5 body bends per minute (BBPM) were removed from the data. All the data were combined and an average calculated and plotted in GraphPad Prism v9. The error bars were calculated as the standard error of the mean (s.e.m.), which is the standard deviation divided by the square root of the number of samples. 4.5. Fluorescent Microscopy Worms of the desired age were mounted on 1% agarose pads in 20 mM sodium azide. Fluorescent imaging was carried out using a Zeiss Imager.M2 microscope at 63× magnification. Images were taken using the Zeiss Zen 2012 Blue Software at 7.4 pixels/µm and representative images compiled using Adobe Photoshop 7.0. Confocal imaging of worms was undertaken on an inverted Zeiss Axio LSM700 equipped with a 488 nm diode laser and LP490 filter. Images were taken at x40 magnification, 1024 × 1024 resolution, with 4× averaging and a Z-stack slice of 1 µm. Stacks were made through the head region of the full animal to encompass all of the fluorescent signal, and then compressed to a maximum projection with Zen Blue 2011 SP3 v8.1 software at 6.4 pixels/µm and compiled in Adobe Photoshop 7.0. 4.6. Quantification of Plaques To quantify the aggregates, ImageJ v1.51 was used, with no additional plugin. Each image was scaled appropriately, the background removed, and a watershed applied. Subsequently, ImageJ supplied the number of aggregates per image and the average size of aggregates in µm2 (calibrated units) was calculated by the ImageJ program. The data were inputted into GraphPad Prism v9 where the averages could be plotted with error bars calculated as the standard error of the mean (s.e.m.). 4.7. Lifespan Assay Worms (strains N2 and NL5901) were age-synchronized and placed on NGM plates containing 100 µM FUdR when at the L4 stage. The plates were maintained at 20 ◦ C throughout the experiment and the worms transferred every 2 days during reproduction to a freshly seeded NGM plate. Worms were assessed daily for death, which was verified when they failed to respond to a gentle tap on the head with a platinum wire. Lifespan was evaluated for worms exposed to Levodopa and Ambroxol, where the same protocol was followed, but in addition to FUdR, the NGM was supplemented with the drug to a final concentration of 1 mM. In all cases, the lifespan was undertaken in 2 independent experiments and combined. Survival curves were generated in GraphPad Prism v9 and analyzed with OASIS 2.0 (Online Application for Survival analysis) [118]. Within OASIS, basic survival analysis was used to calculate the mean/medium lifespan that was assessed using the log rank test (log-rank test) where p < 0.0055 was considered as significant (Bonferroni corrections). 5. Conclusions We further characterized the C. elegans PD model where the disease-causing α-synuclein is expressed in the body wall muscles, exploring the effect of the accumulation of aggregates on lifespan and mobility. We were able to use non-specialist equipment and freely available software to quantify the size and number of plaques, providing an indication as to whether drugs are able to interfere with α-synuclein aggregation, a promising disease-modifying approach for treatment of PD [36,38]. Using this information, we developed an affordable, simple, and effective assay that can be used to explore the effect of possible treatments for PD on multiple hallmarks of the disease. From the selection of compounds, Ambroxol Pharmaceuticals 2022, 15, 512 14 of 19 was identified as highly promising for further studies, which is confirmed by its current status in other trials as a PD treatment. Indeed, the platform does not have to be limited to the exploration of small molecules that impact the disease progression, but could also be applied to other possible therapies including nutrition [119] and exercise [120,121]. We believe that using C. elegans in this drug discovery platform is invaluable for identifying promising candidate small-molecule inhibitors of PD, which can then be prioritized for validation in mammalian models. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ph15050512/s1, Figure S1: The size and number of α-synuclein aggregates from fluorescent microscopy; Figure S2: The effect of a selection of drugs on mobility; Figure S3: The effect of Ambroxol on lifespan; Figure S4: The effect of Ambroxol on α-synuclein aggregates; Figure S5: A DMSO dose response on thrashing and α-synuclein aggregates; Table S1: Description of the mean and maximal lifespan of wild-type and worms expressing the α-synuclein following the different conditions [118,122]; Movie S1: A video to show the body bending of C. elegans and associated counting in ImageJ. Author Contributions: Conceptualization, S.H. and A.M.R.; data curation, S.H., M.v.D., N.K. and D.v.d.K.; formal analysis, S.H. and A.M.R.; investigation, S.H., M.v.D., N.K., D.v.d.K. and A.P.; methodology, S.H., M.v.D., A.P. and A.M.R.; validation, S.H., M.v.D., N.K. and D.v.d.K.; writing— original draft, S.H. and A.M.R.; writing—review and editing, S.H., M.v.D. and A.M.R. All authors have read and agreed to the published version of the manuscript. Funding: The authors gratefully acknowledge funding from the research program VICI with project number VI.C.192.024 and Aspasia (015.015.009) from the Dutch Research Council (NWO) awarded to A.M.R. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data generated during this study are included in this article. Raw data can be provided upon request to the corresponding authors. Acknowledgments: We are grateful to the support provided by the HAN BioCentre, HAN University of Applied Sciences, and would especially like to thank Gijs Maassen and Ambra Miagostovich (HAN BioCentre) for their support on this project. Worm strains were provided by the Caenorhabditis Genetics Centre (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40OD010440). Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. Dorsey, E.R.; Sherer, T.; Okun, M.S.; Bloem, B.R. The emerging evidence of the Parkison pandemic. J. Parkinson’s Dis. 2018, 8, S3–S8. [CrossRef] [PubMed] Dorsey, E.R.; Bloem, B.R. The Parkinson Pandemic—A call to action. JAMA Neurol. 2018, 75, 9–10. [CrossRef] [PubMed] Spillantini, M.G.; Schmidt, M.L.; Lee, V.M.-Y.; Trojanowski, J.Q.; Jakes, R.; Goedert, M. Alpha-synuclein in Lewy bodies. Nature 1997, 388, 839–840. 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Editorial George Dawei Guo University of W es tmins ter It has been four years since our last WPCC Chinese media issue was published. In that time, the People's Republic had gone through a dramatic period, mixed with joy and hope as well as devastation. The year 2008 marked the 30th anniversary of the Deng Xiaoping-Ied Economic Reform. At the same time, it witnessed China's successful Beijing Olympic Games, despite much controversy over the 'typically Chinese' manipulation that characterized the opening ceremony. Last year, the Chinese Communist Party celebrated the 60th anniversary of the founding of the People's Republic; although the most intriguing picture of the celebration was still a magnificent display of military weapons alongside with a massive but highly ordered dancing party held in Tiananmen Square, it seemingly received much more positive coverage within international press than previous celebrations of this kind. In March 2008, a series of riots and demonstrations took place in the Tibetan Autonomous Region and adjacent Tibetan-inhabited areas of the country. The demonstrations not only turned into intensely violent situations in Tibet, but also triggered a global-scale anti-Beijing Olympics row, mainly organized by the Tibetans in exile. Two months after the Tibet incident, on 12 May, in Sichuan province of southwest China, a deadly earthquake that measured at 7.9 on the Richter scale took place, leaving at least 87,000 people dead. This great earthquake immediately made headline news in the global media system; as it had been heavily criticized for preventing the foreign press from freely reporting on the Tibet unrest, the Chinese government provided foreign journalists with much more freedom to cover the earthquake. On 5 July 2009, a series of violent riots over several days took place in Drumqi, the capital of the Xinjiang Uyghur Autonomous Region. The riots started as a protest involving at least 1000 Uyghurs, but later escalated into violent attacks which mainly targeted Han (ethnic Chinese) people. Armed police were deployed to put down the violence and, two days later, hundreds of Han people clashed with both the police and Uyghurs. According to the Chinese government, 197 people died, with 1721 others injured and many vehicles and buildings destroyed in the riots. Uyghur groups, on the other hand, say the death toll was far higher. These 'big Chinese stories' repeatedly remind us that, while its economy is Westminster Papers in Communication and Culture © 2010 (University of Westminster, London), Vol. 7(1): 1-6. ISSN 1744-6708 (print); 1744-6716 (Online) 1 Westminster Papers in Communication and Culture 7(1) growing rapidly, China is also facing tremendous social and political challenges. The term 'risk society' (flngxian shehuz) has become more and more popular for Chinese academics to use in response to all sorts of social or environmental crisis. Although the theory of risk society, which was first introduced by Ulrich Beck in 1986, is widely referenced within Chinese academia, the Chinese intelligentsia seem to have a lack of critical engagement with the theory itself. As far as the Chinese situations are concerned, to use this Western-originated concept uncritically can be quite problematic. In contemporary Chinese society, any kind of social and economic reform is illegitimate unless it is ultimately approved or led by the Communist Party, even though there may exist disagreement and conflict about it among the senior Party officials; the 'partystate' plays a dominant role in controlling and mobilizing political, social and economic resources. Therefore, in the West, especially in the Anglo-Saxon world, the term 'risk society': 'is an important societal conceptualization - risk being a key concept of economics - that also found a political resonance in environmental circles' (Therborn, 2007, 77), whereas in China, it should be primarily associated with the authoritarian state. In other words, whatever sphere a certain risk occupies in China, be it social or environmental, it always poses a fundamental challenge to the legitimacy of Chinese government. Pervasive as it is, party-state intervention is becoming increasingly complex and tactical in China. This is well reflected in the mass communication sphere. After the Tibet unrest, for example, the Chinese government decided to introduce a new rule for dealing with breaking events (ttija shijian). That is, immediately after a particular breaking event occurs, the main body involved in the event should hold a press conference; more importantly, news media should 'report the fact quickly, but be cautious on the causes behind the fact' (kuaijiang shishi) shet!Jiangyuaf!Jin). For some, this 'new' rule can be considered as a step forward, made by the government itself to improve the free flow of information. For others, it is still an old version of news control in a new guise. From this, it can be seen that Chinese media continues to be subjected to a refiguration of party-state power, coexisting with a strong tendency to embrace and sometimes negotiate with an international information order. In the past couple of years, a new corpus of academic work has built up, addressing the latest developments in the Chinese media, with much emphasis on party-state intervention (e.g. Berry, 2009; Sun and Zhao, 2009; Zhao, 2008). Yuezhi Zhao (2008), in her substantial book Communication in China, not only attempts to 'describe the making of authoritarian formations in the Chinese communication system and the role of communication in the constitution of "China's new order" but also [to] analyze the unfolding dynamics of communication politics and the uneven terrains over which various social forces 2 Guo) Editorial struggle for their respective stakes during China's epochal transformation' (p. 10). The book covers a full range of prominent topics mainly concerning the political economy of Chinese media and society, from Chinese media's integration with global capital to the Chinese domestic entertainment TV industry, from civil rights and legal justice to the rise of Chinese neoliberalism. Chris Berry (2009), in his work on Shanghai Television's Documentary Channel, interrogates the Documentary Channel as a specific kind of public space in the culture of Shanghai and China. He argues that Chinese televisual public space exists under the conditions of the party-state apparatus and global marketization. On the one hand, there has been much effort made by the journalists of the Documentary Channel to make the channel a public space using their programming, but the party-state apparatus still 'has ultimate control over what gets aired' and 'the collaboration between the journalists and viewers must fit in with its values' (ibid.: 85). On the other hand, as in the United States and the United Kingdom, as Berry comments, 'the reconfigurations pursuant upon the new multi-channel environment and the increased emphasis on market dynamics and the trading in audience-as-product' have become important factors 'in shaping the ways in which both journalists and viewers can participate in televisual public space as the state' (ibid.: 89). Sun and Zhao (2009), in their provocative article 'Television Culture with "Chinese Characteristics"', take China Central Television's (CCTV) 'emotionally powerful and morally lifting' coverage of the Sichuan earthquake as their starting point (ibid.: 96); they view that kind of 'television storytelling and subject making' as something that 'articulates with the broad political economy of China's ongoing social transformation during the era of a worldwide "neoliberal revolution'" (ibid.: 97). It is the production of the neoliberal subject in the current Chinese sociopolitical climate that they find deeply problematic here. They point out that Chinese television always treats 'human tragedies as either incidental or inevitable', and avoids touching upon 'the social and political causes of individual's suffering' or the flaws of China's social welfare system (ibid.: 104). What they try to argue here to a large extent echoes my argument against an uncritical use of the theory of risk society within Chinese academia; it takes a great deal of empirical effort and in-depth analysis to study the ambiguities and paradoxes of Chinese media in relation to the Chinese state, which is sustained by both socialist legacies and neoliberal strategies. In my opinion, that is also an important goal for a critical Chinese media studies. This special issue of WPCC on media and China wishes to make some contribution to the achievement of that goal. This issue comprises six original articles and one book review. These six articles fall into three categories of topics on media and China. The first two articles deal with two interesting cases of China's communication policy, Digital Rights Management (DRM) on the one hand and the digital television switch over on the other. 3 Westminster Papers in Communication and Culture 7(1) Benjamin Bates and Tao Liu's article considers how China could implement DRM approaches within the international copyright structure that could maximize social value and acceptance. Between the Confucian emphasis on sharing cultural materials and a more politically motivated treatment in both Imperial and Maoist periods, there has been little basis for the intellectual property rights approach favoured by the West and pushed in international trade agreements. Some scholars have questioned whether Western property rights approaches are appropriate for all cultures, and have suggested that DRM systems can offer an alternative approach. Delays in the implementation of copyright procedures and processes in China, particularly in the electronic media, provide an opportunity to develop new approaches. In the article, they argue that China has an opportunity to consider how DRM systems and approaches could be developed to enable an alternative approach to intellectual property rights consistent with traditional cultural values. Michael Starks's article on digital television switchover in China is based on interviews he conducted in the country in 2008, together with good literature research. His article tries to explore why the Chinese route to switchover is different from that of most other countries. He identifies that, elsewhere, the priority has been to switch off analogue terrestrial transmissions to make more efficient use of spectrum, often against a background of satellite and cable growing in importance and a diminishing role for terrestrial reception, but in China, except for Hong Kong, converting analogue cable systems to digital has been the primary switchover activity. He finds that economic and political factors make a marketled policy difficult to design or apply. Moreover, China's motivation is different: rather than pushing to re-use analogue terrestrial spectrum, the government aims to support the interests of the TV-receiver manufacturing industry and, especially, to improve the managed communication of information to the Chinese people. The next three articles bring to the fore the issues of 'autonomy' or 'independence' in China's media practice. Wusan Sun presents a nice case study of media supervision (meiti jiandu) in China. Following up on her article published in the last WPCC Chinese media special issue, Sun continues to critically examine a power struggle between the local and central levels of Chinese political structure through a journalistic event. Her article shows that, in the context of economic and bureaucratic decentralization, central and local governments, as well as central and local media, have respective interests in what is known as 'public opinion supervision' (yulun jiandu). In the specific media supervision event her article studies, every party involved will evaluate their own potential gains and losses, as well as their relationship to the other parties concerned, and make decisions accordingly. The result of such deliberation between the party-state and the media, according to Sun, propels the development of a public sphere in China. She concludes her article by arguing that the development of public Op1nlOn 4 Guo) Editorial superv1slOn 1S a result of strategic alliances among government bodies and the media. Haiyan Wang's article looks at the problem of local press autonomy in China. As Wang states, it is often mentioned that reform of the Chinese media since the 1980s, characterized by the withdrawal of state subsidies, has led to the tension of the media having to serve 'two masters', the ruling party and the market. Yet there has been little examination as to how journalists in local newsrooms operate in this milieu, and how they feel about and deal with various pressures. This article attempts to fill the gap by providing an insider's perspective. It casts some light on autonomy in China's local press, as well as bringing fresh insights to the dynamic between politics, marketplace and media. The article concludes that it is with tactics and guile that journalism at the local level in China struggles, surV1ves, develops and thrives. Moving away from journalistic practices, Rui Liu's article is about the development of independent TV production companies in China. Her article focuses on the evolution of independents and the changing face of the television market. It discusses the ecology of independent television companies in China, and how government regulations are impacting on the TV production market. It argues that independent TV is providing a new face for China's TV market, one often suspected of being imitative, propagandistic and lacking diversity. The last fascinating piece is contributed by Greg Philo, an active figure at the Glasgow Media Group. Different from the rest of the articles within this issue, which generally talk about China on its own, Philo's article focuses on crosscultural perceptions and the processes by which ideas and values move between societies. It is based on focus groups, responses to questionnaires and interviews with 140 Chinese students, cultural workers and teachers. It shows how the experience of living in the UK can both alter prior expectations of the country as well as generating processes of critical reflection about the nature of both China and Western societies. The participants focused on the evaluation of educational systems, the understanding of rights, law and social obligations, and how these affect the current status of women. Social change does not result simply from exposure to new ideas; cross-cultural contacts and experience can also be factors in the movement away from traditional structures, in that they highlight alternative ways of understanding the self in relation to others and new possibilities for social life. This issue's book review is written by Philip Lin, a second-year doctoral student here at CAMRI. As a native of Taiwan, Lin provides a well-grounded understanding of Hsiao-Hung Chang's book Fake Globalization (2007) in the review. From a literature and cultural studies perspective, using a series of 5 Westminster Papers in Communication and Culture 7(1) examples drawn from contemporary audiovisual culture, Chang analyses globalization in four key dimensions - the global visual-image media, the flow of global commodities, the global empire's control, and global popular culture within the Taiwanese context as well as a wider East Asian context. Finally, just to say a few words on the launch of this special issue. The idea for this issue came from the success of our China Media Centre's 2009 conference on the theme of 'Creativity and Innovation in Chinese Media', held on 22-3 June in London. Over 50 scholars and researchers from Europe, the US, Australia, mainland China, Hong Kong and Taiwan attended the conference. Although only two of the conference papers (Bates and Liu; Liu) are included in this issue, I would like to express my sincere gratitude towards all the conference participants who submitted their work. References Beck, U. (1992) Risk Socie!J: Towards a New Moderni!J, originally published in Germany as Risikogesellschaft: At! dem Weg in eine andere Moderne (1986), London: SAGE. Berry, C. (2009) 'Shanghai Television's Documentary Channel: Chinese television as public space', in Y. Zhu and C. Berry (eds.) TV China, Bloomington and Indianapolis: Indiana University Press, pp. 71-89. Sun, W.N. and Y.Z. Zhao (2009) 'Television culture with "Chinese characteristics": the politics of compassion and education', in G. Turner and J. Tay (eds) Television Studies after TV: Understanding Television in the PostBroadcast Era, London: Routledge, pp. 96-104. Therborn, G. (2007) 'After dialectics: radical social theory in a post-communist world', New Lift Review 430an.-Feb.): 63-114. Zhao, Y. Z. (2008) Communication in China: Political Economy) Power and Cmiflict, Lanham, MD: Rowman and Littlefield. 6 

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Multimodal Technologies and Interaction Review Reviewing Simulation Technology: Implications for Workplace Training Diana R. Sanchez 1, *, Amanda Rueda 1 , Kentaro Kawasaki 1 , Saar Van Lysebetten 2 and Daniel Diaz 1 1 2 * Psychology Department, Ethnic Studies & Psychology Building, San Francisco State University, 1600 Holloway Ave, San Francisco, CA 94132, USA Campus UZ Gent, Corneel Heymanslaan 10-Ingang 17, 9000 Gent, Belgium Correspondence: sanchezdianar@sfsu.edu Abstract: Organizations have maintained a commitment to using simulation technology for training purposes because it prepares employees for realistic work scenarios they may encounter and provides a relevant method for teaching hands-on skills. One challenge that simulation technology has faced is the persistent threat of obsolescence, where investment in an up-to-date solution can rapidly become irrelevant in a matter of months or years as technology progresses. This can be particularly challenging for organizations who seek out the best solutions to help develop and train employees while facing the constraints of limited resources and lengthy acquisition times for tools and equipment. Some industries and organizations may benefit from anticipating which technologies might best serve employees and stakeholders in the future. In this manuscript, we took a historical approach, looking at the history of training and the use of simulation-like experiences over time, which helps us identify historical themes in workplace training. Next, we carried out a systematic review of the recent training research using simulation technology to understand how these recent findings help us understand the identified historical themes. Lastly, we summarized the research literature on simulation technology used for training, and highlighted future directions and made recommendations for practitioners and researchers. Keywords: simulation technology; workplace training; workplace preparation Citation: Sanchez, D.R.; Rueda, A.; Kawasaki, K.; Van Lysebetten, S.; Diaz, D. Reviewing Simulation Technology: Implications for Workplace Training. Multimodal Technol. Interact. 2023, 7, 50. https:// doi.org/10.3390/mti7050050 Academic Editor: Deborah Richards Received: 8 April 2023 Revised: 2 May 2023 Accepted: 5 May 2023 Published: 10 May 2023 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/). 1. Introduction Simulation technology lends itself to training and development in the workplace, given the replication of real-world scenarios [1], which allows users to experience real-life situations and grow valuable skill sets while controlling the characteristics and environment of the learning experience [2]. This provides a uniquely valuable tool for organizations, because it offers opportunities not available in other training modalities (e.g., lectures, role plays, computer-based training [3]). For example, early simulation technology for training airplane pilots used ropes and pulleys to replicate turbulence during flight [4]. Simulation technology is evident in virtualized experiences as a way to enhance the realism of the experience [5], in addition to collecting objective metrics, providing immediate feedback, and capturing performance and assessment information [6]. Additionally, simulation technology may help organizations to better prepare employees to enter the workplace [7] due to the increased capability for providing work-related content, hands-on skills, and experiential learning [8,9]. Organizations have prioritized the transfer of usable skills from a training setting into a workforce setting, as seen in 2021 when large organizations reported spending over USD 17.5 million on workplace training despite the resource limitations of the pandemic [10]. Workplace preparation is critical for employees, particularly those who are early in their career. This importance is reflected in large organizational investments in training [11,12]. However, simulation technology is under an ever-present threat of obsolescence [13,14], and there can be concern for the future relevance of an investment when Multimodal Technol. Interact. 2023, 7, 50. https://doi.org/10.3390/mti7050050 https://www.mdpi.com/journal/mti Multimodal Technol. Interact. 2023, 7, 50 2 of 19 considering the adoption of new or upgraded simulation technology for training [7,15]. This creates an understandable need to anticipate the future value of simulation technology for training. In the current manuscript, we attempt to identify a framework for understanding simulation technology by first reviewing the historical progression of workplace training as it has contributed to the development of simulation technology. In this review, we identified historical themes that emerged over time to provide a clearer understanding of the current research literature. Next, we performed a systematic review of recent simulation technology research that has been used for workplace training. From this, we provided a summary of the current research literature to demonstrate the relevance and distinctions of simulation technology and deepen our discussion of training applications. We next discussed the future directions of simulation technology, by discussing the current research streams that may continue to develop over the next several years and may contribute to predicting how simulation technology may advance. 1.1. Historical Themes of Workplace Training Here, we discuss six historical themes of workplace training which contextualize the evolution of simulation technology. We present salient characteristics with respect to workplace training, and provide examples of milestones (i.e., social, scientific, and technological events or innovations) within each theme. The historical themes are generally chronological, but characteristics span time and geography. As such, the discussions and milestones may overlap time periods; see Table 1. We conclude each theme with a discussion of the influence on simulation technology. This historical review provides an interdisciplinary understanding of simulation technology used in workplace training. For a detailed review of workplace training specifically, see [16]. Table 1. Summary of Historical Themes in Workplace Training. Social/Contextual Value of Knowledge Democratization of Knowledge Science of Learning Scaling Productivity Emergence of Knowledge Work • • Birth of philosophy Establishment of first university • Catalyzed desire for knowledge Just-in-time learning • • WWI/II changed workforce needs • Establishment of labor skill levels • Growth of knowledge work Global/dynamic workforce • • Individualized Learning • Adoption of peer-to-peer learning On-the-job training Scientific • • Legal restrictions on research Operationalizing lifelong learning • Renaissance era research • Standardized behavioral research Development of learning principles • Technological • Skilled workforce becomes a competitive advantage • • Invention of printing press Virtualized information sharing • Industrial Revolution standardized work tasks • Formalized workplace research • Yellow Cab Company used one of first simulations • • Job characteristics theory Emphasis on culture/wellbeing • Reliance on information and communication technology • • Shift towards 3rd generation instructional model Use of AI, VR, and computational psychometrics in instructional systems design 1.1.1. Value of Knowledge The first historical theme represents humans’ innate desire for sense-making [17]. The birth of philosophy triggered tensions among intellectual and spiritual leaders, which Multimodal Technol. Interact. 2023, 7, 50 3 of 19 ultimately ignited academic inquiry. Scholars of science, literature, and philosophy in ancient Greece (600–300 BC) were treated as a threat to religious doctrine [18–20]. At a societal level, there were political movements to obstruct scientific study and access to information. This included legislation which restricted the study of anatomy using human cadavers in ancient Rome (753 BC–476 AD; [21,22]). Escalations over the value of intellectual knowledge catalyzed academics’ resolve to seek knowledge. For example, Claudius Galenus (129–217 AD) examined animal cadavers to circumvent legislative restrictions on anatomical science [21]. The historical theme therefore exemplifies the consequences of intellectual curiosity, and the high stakes of learning. The value and stakes of learning may be reflected in the competitive advantage of employing a knowledgeable and skilled workforce. Workplace training is considered a benefit to individual employees which could contribute to their advancement or promotion [23]. Training researchers have advocated for the integration of lifelong learning into talent development, which creates informal, intentional, and self-directed opportunities for learning [24]. Employees also demonstrate greater interest in companies with strong learning economies that promote their development over time [25], leading organizations to seek innovative ways to keep employees engaged in learning. Simulation technology is one method consistently utilized by organizations to expand their learning opportunities for employees [26,27]. 1.1.2. Democratization of Knowledge The increased value of knowledge led to an inevitable era of increasing the dissemination of knowledge. Technological developments such as the printing press during the Renaissance (1300–1700) increased public accessibility to information. Spiritual advocates sought to re-establish influence by controlling information, such as book bans from the Roman Catholic Church when content was perceived as heretical or lascivious [28]. Attempts at censorship engendered the democratization of knowledge. The Renaissance promoted the open pursuit of intellectual information (e.g., Leonardo da Vinci, 1452–1519, Nicolaus Copernicus 1473–1543, Galileo Galilei, 1564–1642), and the mass distribution of information contributed to a social shift from knowledge being exclusive to scholars, to being shared with the public. The continued pursuit of democratized knowledge has influenced innovative systems for sharing information. Digitization has simplified the means of documenting, maintaining, and disseminating knowledge in organizations. For example, learning management systems and other digital platforms can lower the cost of traditional training programs (i.e., synchronous, in-person; [29]). Modern calls for equity have shaped continued efforts to make knowledge accessible, with many organizations placing workplace training curricula in open access repositories for employees [30]. By making content readily accessible, organizations may optimize when and how knowledge is provided, providing learners with relevant support when they need it [31]. Simulation technology can also provide just-in-time training solutions with immersive, hands-on experiences available to practice virtually for learners when needed. In the healthcare industry, simulations have been used to develop skills such as clinical decision-making, assessing risks, and refresher training on surgical procedures. Further, simulations used in conjunction with pre-briefing and repeated scenarios have been linked to improved learning outcomes for nurses [32]. 1.1.3. Science of Learning Interest in knowledge acquisition is evidenced by prolific research in human memory and pedagogy [33–36]. Formalized research on the mechanisms of learning have brought learning to the forefront of scientific interest. With the standardization of scientific methods [37], researchers examined new facets of learning including the mental processes associated with memory [38,39] and concepts such as observational learning and classical conditioning [40,41], which inform underlying principles used in workplace training today. Multimodal Technol. Interact. 2023, 7, 50 4 of 19 The fundamental concepts developed during the time of formalized research in learning and pedagogy have informed the primary frameworks, theories, and principles applied in formal workplace training today. For example, learner control is a broad term for various instructional design techniques and may be effective for learning through repeated practice [42]. Learner control is one stream of research which has influenced an early adoption of computer-based and simulation technology used for training [43]. Technology-based training is ubiquitous in today’s workplace training environments, and good learning programs are based on robust, scientific learning principles (e.g., immediate feedback, practice opportunities) [14]. 1.1.4. Scaling Productivity A salient theme in modern history has been a focus on maximizing productivity through the application of learned knowledge, skills, and behaviors relevant to the job [34]. This has been important for organizations aiming to improve work-related skills through formalized training programs. One of the earliest pioneers of labor skills was Adam Smith [44], who introduced classifications of labor (common, skilled, qualified) which were characterized by the complexity of skills involved with the tasks. A need for efficient workers (i.e., WWI, the Industrial Revolution) [45] and burgeoning scientific interest in studying the world of work (Frederick Winslow Taylor, 1856–1915, Munsterberg, 1863–1916, Lillian Gilbreth, 1878–1972) created a fertile environment to study large-scale productivity in the workplace [18,46,47]. Although there was some decline in progress during the Great Depression [48], as jobs became increasingly complex and dangerous, organizations found new ways to assess and train workers. For example, in 1925 the Yellow Cab Company in Pittsburg commissioned one of the first uses of simulation technology for training to assess applicants on switchboard reaction time [49]. Interest in simulation technology for training was renewed during WWII due to the high-stakes nature of military training [50,51]. With the emphasis on productivity, a realization emerged that traditional training methods were not always sufficient for intricate task work, and simulation technology presented a viable alternative because it can create levels of complexity which mirror the real world. For example, officers can be placed in a hostage situation where they need to both verbally negotiate and physically react to a simulation of a perpetrator projected on a screen [52]. Firefighters may need to practice navigating a burning building, surveying safety risks, and completing tasks with limited visibility from equipment and smoke [53]. Simulating these complex conditions in replicated environments that can be customized to the unique needs of the learner allows intricate learning to take place within the context of safe, controlled environments [54]. 1.1.5. Emergence of Knowledge Work In addition to the growing complexity of work-related tasks, the emergence of knowledge work has placed technology at the center of the workforce and organizations expect employees to have a basic level of skill in using information and communication technologies (ICTs) [55]. Knowledge work encapsulates types of jobs in which cognitive processes (rather than physical tasks), autonomy, and ICTs are fundamental to performance. Knowledge work has shifted perspectives on technology, moving from technology as a perceived means for optimizing work to regarding technology as a fundamental tool for completing work. The ever-increasing globalization of work has ushered in a new conceptualization of where, when, and how individuals work. Flexible work locations and instant access to information is increasingly present in dynamic work environments [55,56]. Frequent changes in organizational structure (e.g., mergers, downsizing) and unpredictable environments require adaptability from both employees (e.g., being adaptable and resilient) [11] and employers (e.g., evolving workplace practices and services to employees). Employees expect more from workplace culture in terms of the experience they have at work and the degree of support they receive for their wellbeing. Reference [57] suggested that job-related factors influence an individual’s psychological states (e.g., well-being), which in turn plays Multimodal Technol. Interact. 2023, 7, 50 5 of 19 a role in their job performance, degree of satisfaction, and work motivation. Workplace training is one way that organizations can develop the knowledge and skills which may help employees feel an increased sense of autonomy, reduce ambiguity and conflict in their role, and improve manager feedback practices. Simulation technology has been advanced by two factors related to the emergence of knowledge work: the increasing availability of advanced tools, and the globalization of the modern workforce. The growing development and accessibility of tools (i.e., asynchronous communication and collaboration platforms, company intranets) has made simulation technology easier and more affordable for institutions to implement [58]. This offers a potential efficiency gain for organizations, particularly when compared to the financial costs and time costs associated with bringing together geographically dispersed teams for traditional, in-person training solutions. Ultimately, advancements in simulation technology have been accelerated by innovative technologies that allow organizations to remain competitive. 1.1.6. Individualized Learning The shifting nature of work has resulted in employees expecting individualized care, attention, and resources provided by their organization [59]. For example, there is an increased desire for and availability of one-on-one experiences such as mentoring and coaching resources for companies [60]. Mentoring offers an individualized learning experience in which experienced professionals offer guidance and support to novice employees [61]. Executive coaching is a type of leadership development that is characterized by a high contextual-sensitivity coaching approach due to the complex roles and relationships among executives’ stakeholders [62]. Despite the benefits of these one-on-one methods, they can come at a high cost [63]. An alternative method that organizations and institutions have tried implementing is alternative peer-to-peer learning experiences, which may take the form of on-the-job training wherein a novice and an expert are paired together [64]. Although the individual in the expert role may have limited motivation, skills, or knowledge, learners tend to enjoy the individualized attention in these interactions [65]. Although these are not new learning modalities, they have been facilitated by the increasing availability of virtualized learning platforms [66]. This shift from instructor-based learning to interaction-based learning (i.e., third-generation instruction) has challenged researchers and practitioners to rethink how training methods support learning [66]. Interaction-based learning takes a social constructivist view and highlights the importance of the learning environment and social context where learners drive their experience, thus facilitating individualized learning to a higher degree because of the unique set of knowledge, skills, and abilities that each learner chooses to engage in. With a shift towards individualized experiences, simulation technology can be highlighted as it can provide individualized learning experiences at scale across large groups of geographically dispersed individuals [1]. The automation and customization of simulation technology can include collecting objective metrics on learners and feedback mechanisms (i.e., scoring features) customized to a user, or detailed and customized opportunities to practice. These features exceed what may be possible from a human instructor. The primary drawback to simulation technology for workplace training is the upfront cost and expertise required to design the programming needed to leverage these advanced technologies appropriately (e.g., [67]). A summary of historical themes is provided in Table 1. 2. Method Current Research on Training Simulation Technology In this section, we provide a systematic review of the current research literature on simulation technology that has been used for workplace training in order to prepare people for the skills and tasks required on-the-job. The following criteria were used to select papers: • Peer-reviewed journal articles published since the year 2000. Multimodal Technol. Interact. 2023, 7, 50 6 of 19 • • Samples of working-age individuals between ages 18 and 65. Methodology had to include simulation technology which was intended to prepare individuals for work. This means that the simulation technology needed to replicate a real-world work environment for the purpose of training an individual. Had to include a virtualized component. • The methodology inclusion criteria were particularly important as the current paper aims to provide an overview of current simulation technology used for workplace training. Research papers which used participants that would be subject to specific employment laws and regulations (e.g., protected classes, neurodiverse populations, etc.) were excluded as this study aims to provide a review of simulation technology in standardized workplace training contexts. Papers were identified through searches of the following electronic databases: Academic Search Ultimate, APA PsychInfo, Applied Science & Technology Source, Academic Search Premier, and Business Source Premier. The most recent search was performed in April 2023 using the following boolean search terms: Title/Abstract contains (“simulation technology” OR “simulation training” OR “work simulation” OR “workplace simulation” OR “job simulation”) AND (“workplace training” OR “workplace learning” OR “job training” OR “job learning” OR “organization* training” OR “organization* learning”). 3. Results 3.1. Summary The result initially provided us with 64 articles. We filtered those according to the criteria above and found 18 papers that met our requirements. This final selection of papers covered the use of simulation technology intended to develop work-relevant skills and abilities. The literature review is categorized in Table 2 according to the simulation type, subject matter, and outcome measures. Table 2. Overview of existing simulation technology literature. Article Year Simulation Type Subject Matter Outcome Measures Sample Participants [68] 2005 Computer-driven simulation Medical Job-related task performance specific to interrupted suture score 11 Postgraduate Medical Residents [69] 2005 Virtual Reality-based dental training Dental Job-related task performance 42 Dental Students [70] 2006 Virtual Reality training simulator Medical Job-related task performance specific to carotid angiography 20 Interventional Cardiologists 29 Space Shuttle MMT Members [71] 2007 Aerospace simulator Aerospace Job-related task performance, Communication, Teamwork and Reactions [72] 2009 Web-based work simulation Business Performance as moderated by self-reflection 360 Employees [73] 2012 Computer-based training simulator for industrial machinery Construction Reactions 56 Participants [74] 2012 Virtual Reality simulation with haptic properties Medical Job-relevant skills and reactions 10 Undergraduate Students Multimodal Technol. Interact. 2023, 7, 50 7 of 19 Table 2. Cont. Article Year Simulation Type Subject Matter Outcome Measures Sample Participants [75] 2013 Businesssimulation computer game Business and Leadership Reactions to leadership behaviors 26 Graduate Students [76] 2015 Non-interactive 10 min 3D video Medical Empathy (modified Kiersma–Chen empathy scale) 460 Undergraduate Nursing Students [77] 2015 High-technology human patient simulator (HPS) Medical Patient assessment skills 101 Undergraduate Students [78] 2016 Safety and security training simulator for ship handling Public Safety Job-related performance 14 Students [79] 2016 Flight simulator Aerospace Job-related task performance in flight abilities 29 Students [80] 2017 Medical manikin Medical Reactions 17 Doctoral Students 2019 Healthcare interactive virtual simulation training system (HH-VSTS) Medical Job-related task performance specific to hazard management 74 Healthcare Workers and Students [82] 2019 Maritime education and training (MET) Simulator Maritime Confidence in task performance 11 Students [83] 2019 Medical manikins Medical Comfort with medical interventions 57 General Medicine Officers [81] [84] [85] 2020 Medical manikin Medical Interteam communication ~26 Four Neurosurgeon teams; four medical student teams 2020 Experiential gamified simulation (WAGESBusiness) Business Acknowledgement of unconscious bias 126 Undergraduate Students As is evidenced by the findings of our systematic literature review, simulations are used primarily for complex skills and tasks. Below is a general summary of the articles we found. The articles spanned publication from 2005 to 2020. Ten of the articles focused on dental or medical procedures [68–70,74,76,77,80,81,83,84]. Thirteen of the articles used students or recent graduates at least in part for their sample [68,69,74–82,84,85]. Ten of the studies directly measured job-related skills or tasks in some way as an outcome measure [68–72,74,77–79,81]. Five measured reactions to the simulation as part of their outcomes. Based on our review of this literature, we provide first a summary distinguishing different types of training technology, followed by an overview of the identified benefits and challenges of using simulation technology. Multimodal Technol. Interact. 2023, 7, 50 8 of 19 A Summary of Current Simulation Technology What we found in our reading of the current research was that as the workplace training landscape continues to evolve, simulation technology has transitioned from primarily manual mechanisms [4] to synthetic learning environments [3]. Within synthetic learning environments, simulation technology has emerged as a training medium that creates, extends, and manages learning objectives [86]. Simulation technology advances classic observational learning in simulations by aiding realism and embedded instruction [40,86]. Thus, simulation technology enables learners to interact with multi-faceted, complex issues where they can apply prior knowledge and skills to real-world issues related to their discipline [87]. 3.2. Similarities and Distinctions from Related Training Approaches Overlapping conceptualizations of training methods such as games or roleplays are common in workplace training research [6,88]. The application of simulation technology in workplace training can be distinguished by the degree of realism (i.e., fidelity) embedded into the design and delivery [81,89]. Additionally, simulation technology used in workplace training consistently takes place in scenario-based environments, where individuals interact with the environment to apply prior knowledge and practical skills [87,90,91]. Although simulation technology used for workplace training is distinct from other training methods, elements of synthetic and scenario-based learning environments can be used with many delivery methods. We offer a brief overview of training methods commonly addressed with simulation technology for workplace training in overlapping terms: serious games, gamebased learning, and role plays. Table 3 highlights similarities and distinctions between these terms. Table 3 is not an exhaustive overview, but is intended to illustrate where overlap occurs in these training modalities. Table 3. Overview of Terminology Related to Training Simulations. Simulation Learning Objectives Instructional Principles • • • • • • • • • • • Serious Games Role Play • • • • • • • • • Transfer Self-Efficacy Teamwork Skills Procedural Knowledge Motivation Communication Skills Perceptual Knowledge Psychomotor Skills Transfer Self-Efficacy Teamwork Skills Procedural Knowledge Motivation Declarative Knowledge Transfer Self-Efficacy Communication Skills Design Attributes Commonly Used Context Scaffolding 2 Repetition 4 Motivating Learners 3 Variability of Roles, Responsibilities, Strategies, etc. 1 Error Management 1 Adaptive Difficulty 2 • • • • • • Immersion t Rules/Goals t High Fidelity Scenario-Based Gamification * Gameful Design * • • Scaffolding 2 Motivating Learners 3 Metacognitive Prompts 3 Contiguity 5 Pretraining 5 • • • • • • • • • • • Action Language t Assessment t Conflict/Challenge t Control t Environment t Game Fiction t Human Interaction t Immersion t Rules/Goals t Gamification * Gameful Design * • • • • • • • Education Business Mathematics Science Technology Healthcare Business • • Scaffolding 2 Repetition 4 • • • Scenario-Based Gamification * Gameful Design * • • • Business Language Leadership • • • • • • • • • • • Healthcare Engineering Aerospace Law Enforcement Physics Delivery Media • • • • • • • Face-to-Face Computerbased Web-based Virtual Reality Augmented Reality Mixed Reality Wearable Tech Note: Bolded values are more salient to the given methodology. t [26] 1 —Effective Practice; 2 —Optimize Sequencing; 3 —Engage learners in their own learning; 4 —Develop past initial mastery; 5 —Organize content (Adapted from previous research [16]). * Gamification and gameful design employ one or more game design attributes to develop game-based learning interventions, whereas serious games consistently use all game design attributes, albeit in different ways or to varying degrees. Multimodal Technol. Interact. 2023, 7, 50 9 of 19 3.2.1. Serious Games Serious games are games used specifically for educational rather than entertainment purposes [88]. Conversely, simulation technology may be used for non-educational purposes such as pure games (e.g., [92]) and economic simulations [93]. Beyond purpose, simulation technology and serious games can use game attributes differently in their design [26,27]. Serious games employ a complete set of attributes associated with game development, whereas simulation technology for training may use a subset of game attributes without taking the form of a game [75,88]. For example, both serious games and simulation technology for training include clear rules and goals of the mission to be achieved [26,94]. Similarly, the game attribute of immersion enables complex technical and social interactions that would occur in real-life to take place within both games and simulation technology used for training [95]. For example, a military game would likely use the context of basic exercise drills or missions [83] rather than an office setting to create an immersive environment. 3.2.2. Game-Based Learning Game-based learning is the use of game-based technology to deliver, support, and enhance teaching, learning, assessment, and evaluation [96]. Unlike serious games (in which the game delivers the learning content), in game-based learning the instructor remains the primary provider of learning content and uses game elements to increase learner motivation [88]. To this end, instructional designers may employ different strategies to embed game-like elements into learning content or delivery methods, including simulation technology. One strategy, gamification, involves incorporating specific game attributes into existing training, typically to improve learner engagement [95]. Similarly, gameful design places game attributes at the center of the development process for new training or assessment instruments [97]. A game can be set within the context of a simulation depending on how the environment is designed. 3.2.3. Role Plays In role playing, individuals play pre-determined characters and invent the scenario in real-time, whereas simulation technology represents systems in which participants have functions [98]. Role plays are an efficient training method when trainers want to focus on specific training content and evaluate learner performance in a particular skill [76,99]. One potential explanation for the efficacy of role plays is attributed to the social and observational learning that can occur as a participant or an active spectator. Role plays accelerate how quickly learners integrate new knowledge and skills into behaviors [100]. Thus, a primary similarity between role plays and simulation technology used in workplace training is the scenario-based learning environment which challenges learners to apply their knowledge. A roleplay can be set within the context of a simulation depending on how the experience is designed. 3.3. Benefits of Simulation Technology With respect to learning outcomes, we adopted the view of researchers who have asserted that instructional principles are more impactful than instructional mediums (i.e., computer, paper, video, audio) [16,101]. This is an important point in the discussion of potential benefits of simulation technology for training because the media, like the design, should be a function of the learning objective [102]. To illustrate this relationship, we explored how simulation technology facilitates various instructional principles. 3.3.1. Realism The realistic nature of simulation technology is a salient feature and beneficial to learning outcomes [74]. Depending on the content and purpose of the training, simulation technology can provide physical fidelity (i.e., similarity in look, sound, and feel) and psychological fidelity (i.e., similarity in mental and/or emotional state) to facilitate learn- Multimodal Technol. Interact. 2023, 7, 50 10 of 19 ing [103]. The potential for high fidelity in simulation technology is critical to its efficacy as a learning medium because it bolsters retention via identical elements (i.e., the similarity between the training and the real-world application; [16,104]. Research on training transfer has suggested that skills are more likely to be reproduced in work environments that more closely resemble the training environment [67,105,106]. Simulation technology for workplace training enables instructors to create practice scenarios for nuanced behavioral skills, such as the stress management and adaptability required for empathic communication [76,107]. Given the traditional use of in-person instruction and the common use of computer-based training [53], simulation technology may help to bridge this gap by developing users’ skills for lifelong learning outside of a formal learning environment. For learners using simulation technology, this can be considered an additional element of fidelity beyond focal knowledge or skill. 3.3.2. Practice Opportunities Simulation technology for training can afford learners with opportunities to exercise control over their practice [77]. Control in simulation technology has been characterized as a learner’s decision latitude over their strategies, role, and assumed responsibilities [6]. A constructivist view of learning suggests that using different vantage points to problem solve, create meaning, and apply knowledge may contribute to the creation of mental models [66]. High control simulation technology affords learners the latitude to isolate and repeatedly practice a skill, which can be considered effective practice [16]. Further, learner control over difficulty has been shown to have a strong moderating effect between self-efficacy and learning transfer in training which uses simulation technology [5]. In digital training settings, learner control and adaptive guidance have been shown to be effective design elements for complex skills by allowing learners to build on previous or fundamental knowledge [107]. Indeed, repeatedly practicing decision making in various roles and learning from mistakes have been reported as critical deep learning elements by learners using simulation technology [42]. 3.3.3. Immersive Environments Practicing using simulation technology is advantageous when a real-life environment is high stakes [5], meaning the tasks could be highly complex, have small margins of error, or present potentially dangerous or intimidating experiences [13,108]. The immersive elements of simulation technology used for training have been positively linked to effective learning procedures [108], which promotes simulation technology as an alternative to training scenarios where a mistake could result in serious injury, death, damaged equipment or other losses. For example, intimidating environments can add a degree of psychological stress to a work task [103]. Construction workers on a high-rise worksite need to demonstrate familiarity and comfort with safety procedures and tasks at a great height before being expected to do so in real life. Ultimately, utilizing simulation technology for training offers affordance to engage in error management training without the consequences of actual physical, emotional, or economic harm [82,109]. 3.3.4. Feedback Capabilities Simulation technology might be able to provide performance improvement feedback via behavioral metrics [6,14]. Detailed feedback can be integrated into simulation technology using techniques such as debriefing and metacognitive prompts, which have historically been linked to training transfer [27,43,72,110]. Developments in the field of computational psychometrics suggest that trace data (i.e., mouse movements/clicks, time spent on a task, selection choices) can be mined to predict individuals’ cognitive ability or personality traits [111]. This preliminary evidence highlights the variety of data available to practitioners for use in providing mid- and post-training feedback. Additionally, learner metacognition has been highlighted as a fundamental state in the active learning pro- Multimodal Technol. Interact. 2023, 7, 50 11 of 19 cess [43]. In a review of simulation technology [6], the authors suggests that metacognitive interventions may be critical for knowledge outcomes in particular. 3.3.5. Cost as an Investment Utilizing simulation technology for training may also provide long-term cost savings, despite the high upfront cost typically associated with well-designed solutions [54,79]. The flexible yet controlled environment of simulation technology allows organizations to develop standardized and customizable training programs that can mitigate the potential downsides of on-the-job training, such as untrained handling of equipment, premature execution of tasks by untrained or unskilled personnel, and potential worksite dangers and hazards which could lead to worker’s compensation claims [2,112]. Thus, when evaluating the upfront cost of simulation technology as a training solution, it can be valuable to consider the opportunity for subsequent long-term cost efficiencies. In addition to the reduced maintenance costs, [113] found that one particular intervention, using simulation technology, produced cost savings by reducing administrative costs such as longer hospital stays. The costs of developing simulation technology for training can also be offset by overlaying innovative solutions such as augmented reality with existing training tools (e.g., medical manikins) or lower cost interfaces (e.g., tablets) [58,83]. Additionally, the skills required to develop simulation technology (i.e., coding, engineering, etc.) are becoming more available as STEM fields continue to grow [114]. The increasing availability of skilled programmers and product designers works to make simulation technology more accessible and affordable to organizations. 3.4. Challenges with Simulation Technology Here, we highlight pertinent challenges to be aware of when considering using simulation technology for training. 3.4.1. Upfront Costs There is a relatively high upfront cost associated with some simulation technology, usually associated with the time, expertise, testing, and revising involved in the development process. These costs can pose a financial risk if organizations do not realize the expected participation. Preliminary findings have suggested that a viable alternative to developing custom game-based interventions is to use commercially available off-the-shelf games, which are typically less resource intensive [54]. Since game-based learning shares attributes with simulation technology, it follows that commercial options may emerge as a path for simulations. 3.4.2. Variability in Learner Experiences Secondly, there is variability in the applied knowledge, skills, and abilities for similar jobs and tasks. The breadth and ambiguity of learner tasks may create challenges for data collection in simulation technology [115]. This variability could result in a more resourceintensive process for instructional designers, workplace trainers, and educators since the efficacy of simulation technology as a training solution relies heavily on recreating real-life scenarios. To avoid this pitfall, organizations can consider using simulation technology for training generalizable skills versus specialized skills. For example, training on a forklift can be generalized based on the most common conditions (e.g., warehouse, side-loading, rough terrain, etc.), regardless of the specific forklift manufacturer. However, generalizable skills may still vary between individuals and the outcome of using simulation technology could produce results that would be difficult to mimic in everyday life. 4. Discussion 4.1. Modern Research Findings Here, we provide insights on emerging research to reduce the challenges of rapid advancements. While this is not an exhaustive review, we did attempt to provide an Multimodal Technol. Interact. 2023, 7, 50 12 of 19 overview of recent research literature. The three themes we highlighted include design attributes, pedagogical challenges, and cognitive load effects (Table 4). These themes, drawn from the research review described above, extend the historical themes from Table 1. Table 4. Linking Modern Themes in Simulation Technology with Historical Themes in Workplace Training. Science of Learning: Establishment of parsimony has been an effective accelerator for similar training method research (e.g., serious games, game-based learning). Design Attributes Democratization of Knowledge: Availability of interdisciplinary research may lead to novel design attributes. Methodological studies are also critical to isolate and manipulate individual attributes. Scaling Productivity: Future research should explore design attributes which facilitate incremental observational and social learning compared to traditional methods. Individualized Learning: A taxonomy of simulation design attributes would enable educators to design interventions that take into account learner characteristics. Emergence of Knowledge Work: Future research should continue to study the effects of cognitive load on simulation training transfer to improve its design efficacy. Democratization of Knowledge: Examining cognitive load effects on learning via simulation for training can mitigate potential adverse impacts to neurodiverse learner populations. Cognitive Load Scaling Productivity: Simulations may simplify learning by reducing specific types of cognitive load compared to natural environments. Individualized Learning: All feedback is not created equal, especially in simulations with high fidelity. Research focused on the cognitive load effects of various feedback mechanisms will ensure simulations are designed effectively. Science of Learning: Industrial training effectiveness research may offer frameworks for building digital competencies. Additionally, higher learning institutions may realize additional benefits, such as educator job satisfaction and retention. Pedagogical Challenges Democratization of Knowledge: Pedagogical research will enable increased access to simulation-based learning and educator capabilities. Scaling Productivity/Individualized Learning: Simulation for training is not a substitute for traditional methods in most cases. Educators play a key role in adding feedback and meaning to learners’ simulated experiences. 4.1.1. Design Attributes Researchers continue to reinforce the role of intentional design, methodology, and media selection to effectively facilitate learning objectives (e.g., [54,72,102]). Recent research has built upon existing instructional systems’ design taxonomies and established theoretical implications for simulation technology design attributes in the context of training (e.g., [13,116,117]). For example, previous research has explored differences between gesture-based motions (i.e., using the thumb and index finger to zoom in/out of a screen) and mouse-based motions (i.e., clicking an icon which represents zoom) [118]. The authors found that young learners in the multi-touch, gesture-based group spent more time interacting with the learning content, likely due to the intuitive nature of the gesture-based motions. This is just one example of how modern advancements create room for nextgeneration design attributes in simulation technology. Delineating training design elements for use specifically with simulation technology parallels efforts to establish parsimony within the game-based learning literature. We expect to see more research on instructional design for simulation technology and encourage practitioners to familiarize themselves with emerging taxonomies. 4.1.2. Cognitive Load Recent research in the medical field suggests growing interest in the linkages between cognitive load and learning outcomes in simulation technology used for training (e.g., [27,32,119–121]). The interaction of cognitive load in a learning environment using simulation technology is applicable beyond medicine. Previous research has found that Multimodal Technol. Interact. 2023, 7, 50 13 of 19 in-game metrics predicted cognitive load (and subsequently performance) within the first tenth of the game time [122]. This preliminary research is promising for the development of adaptive features associated with simulation technology in service of maximizing challenges while mitigating negative effects of overload. Similarly, other researchers have studied the effects of text annotation on cognitive load in the use of virtualized simulation technology [120]. In contrast to the cognitive theory of multimedia learning [123], the text annotations did not significantly reduce extraneous cognitive load. This finding was explained by the simple visual display of the learning material, such that learners did not need to divert cognitive resources to learning what had been provided in the display. We expect researchers and developers to continue narrowing in on how to leverage the benefits of simulation technology, including a potential for more deep learning and less cognitive load unrelated to the learning. 4.1.3. Pedagogical Challenges Beyond the medical field [7,15], there is a lack of empirical guidance for integrating simulation technology into workplace training, which could slow work-readiness for those entering a workforce. However, there have been calls for increased digital competencies to enable organizations to meet continuously evolving demands. For example, the mass adoption of virtual training solutions challenged organizations to learn and execute technology-based training solutions. Industrially, there has been a paradigm shift from using digital tools for efficiency to using digital tools as an everyday necessity. International governments and organizations have begun to highlight specific digital competencies such as media literacy and digital content creation skills (i.e., programming), even addressing these as a key to economic growth [7]. In one previous research study, the authors present a framework which applies instructional design components to the requisite knowledge, skills, and abilities associated with using simulation technology in healthcare (i.e., simulation design, scenario design, simulation research, simulation program administration) [119]. Pedagogically, there appears to be consistent support that simulations are likely to be effective when used as a supplement to traditional instructional techniques (e.g., [6,120]). 4.2. Recommended Use of Simulation Technology Based on the present review, we offer a direction on learning situations in which we recommend simulation technology. Here, we draw from previous researchers who mapped simulation technology learning outcomes to address specific behavioral, affective, and cognitive outcomes [6]. We add to this work by offering supportive empirical and theoretical research from the field of workplace training. Simulation technology has been shown to have a positive relationship with behavioral outcomes such as perceptual motor skills, teamwork, and social skills [6]. We strongly recommend using simulation technology for behavioral learning outcomes due to the transfer benefits associated with high fidelity learning environments [16,104,105]. Research suggests that training fidelity is critical when a skill is highly complex (e.g., self-regulation of cardiovascular response) [109] or when the physical environment is highly sensitive (e.g., operating a military aircraft) [4]. Additionally, simulation technology may be especially well-suited for affective learning outcomes such as motivation, self-efficacy, and training satisfaction [6,82]. We suggest using simulation technology for affective outcomes when practitioners have access to simulation technology solutions which evoke immersive experiences and afford learner control over the difficulty of the task. This recommendation may have a heightened impact based on meta-analytic evidence suggesting that learner control enhances the relationship between self-efficacy and transfer [5]. Finally, simulation technology has been shown to be particularly effective for advanced cognitive skills such as critical thinking, decision-making, and meta-cognitive learning strategies [53]. Thus, we recommend that educators consider utilizing simulation technology for training when a high-fidelity environment is requisite to the focal knowledge (e.g., physics, engineering). For example, simulation technology has consistently been shown to effectively develop spatial reasoning (e.g., [118,124,125]). Similarly, simulation Multimodal Technol. Interact. 2023, 7, 50 14 of 19 technology may be effective for procedural knowledge, rather than declarative knowledge, as a result of mental models’ construction [120,126]. Beyond these use cases, simulation technology may not be a cost-effective training methodology for other cognitive outcomes. 4.3. Limitations While this paper provided informed predictions about the future of simulation technology for training, we acknowledge that cutting-edge science in this field is rapidly advancing. This review offers a snapshot of the intersection of simulation technology and workplace training, but readers are advised to seek out the most recent research and use cases. We hope future empirical research on the application of simulation technology will continue to acknowledge relevant advancements in the field as a way to continuously bridge the gap between reviews. For practitioners, we also recognize that the pace of innovation and implementation may often outpace the pace of science. We hope the historical review provided in this paper provides context for evaluating the new features of simulation technology as they become available. 5. Conclusions Simulation technology has emerged and evolved in a way that aligns with the advancing needs of training interventions in the workplace. From manual mechanisms to synthetic learning environments, simulation technology likely enables learners to interact with complex issues and apply their skills to real-world scenarios related to their discipline. While simulation technology used in workplace training shares similarities with serious games, game-based learning, and role plays in terms of using scenario-based learning environments, it differs in the degree of realism embedded in the design and delivery. Understanding these similarities and differences may help organizations develop a well-rounded training strategy which uses multiple modalities to achieve varying learning outcomes. We also discussed the importance and application of evidence-based instructional principles when designing training using simulation technology. Integrating effective workplace training practices is critical for organizations seeking to reap the benefits of simulation technology, which include the improved transfer effects of immersive environments, high levels of learner control, and in situ feedback capabilities. Designing simulation technology for workplace training with instructional principles and a clear strategy may also provide longterm cost savings despite the high upfront cost of development. These points offer important insights into the potential knowledge, skills, and capabilities needed by professionals in learning and development, instructional design, or any other education-related role Finally, emerging research was presented in an effort to equip practitioners with informed predictions to help reduce the challenging effects of rapid advancements in simulation technology. We highlighted three themes we expect to see more research on in the near future: design attributes of simulation technology for workplace training, cognitive load effects of using simulation technology, and increasing urgency for pedagogical support in higher education. Finally, we recommended using simulation technology for training primarily when a high-fidelity practice environment poses potential imminent risk to the learner, as well as for complex cognitive tasks and knowledge development. Author Contributions: Conceptualization, D.R.S.; methodology, D.R.S., S.V.L. and A.R.; software, D.R.S., A.R. and K.K.; validation, D.R.S., A.R. and K.K.; formal analysis, D.R.S., A.R. and K.K.; investigation, D.R.S., A.R., K.K. and D.D.; resources, D.R.S., A.R., S.V.L., K.K. and D.D.; data curation, D.R.S., A.R. and K.K.; writing—original draft preparation, D.R.S., A.R. and S.V.L.; writing—review and editing, D.R.S., A.R., K.K. and D.D.; visualization, D.R.S., A.R. and K.K.; supervision, D.R.S.; project administration, D.R.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Multimodal Technol. Interact. 2023, 7, 50 15 of 19 Data Availability Statement: All data generated or analyzed during this study are included in this published article and its supplementary information files. 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W3215581205.txt

LITE: Jurnal Bahasa, Sastra, dan Budaya ISSN online: 2548-9588 ISSN print.: 1907-3607 http://publikasi.dinus.ac.id/index.php/lite Published by Universitas Dian Nuswantoro, Semarang Techniques for Absorption into Indonesian and the Acceptance of Loan Words Related to Covid-19: Socioterminology Approach Lalu Muhamad Helmi1, Mahsun Mahsun2, Burhanuddin Burhanuddin3 Universitas Mataram, West Nusa Tenggara, Indonesia Universitas Mataram, West Nusa Tenggara, Indonesia Universitas Mataram, West Nusa Tenggara, Indonesia Article History Abstract Submitted date: 2021-06-17 Accepted date: 2021-09-21 Published date: 2021-11-28 Keywords: This study answers two questions: what equivalence rules of terms are dominantly found in the absorption words during the Covid-19 pandemic, and how acceptable these terms are in the Indonesian-speaking community. The data were collected by using documentation or observation method, supported by the use of a questionnaire. The data were analyzed by using intralingual equivalence and descriptive analysis methods. The results show that 33 foreign terms were matched using translation techniques, ten words with absorption techniques, and two words with combined translation and absorption techniques. Furthermore, the acceptability of absorption terms during the Corona virus pandemic is 39%, while 61% indicates the unacceptability of absorption terms during the Corona virus pandemic. terms, Covid-19, socioterminological approach, matching rules, acceptance of terms Abstrak Kata Kunci: istilah, Covid-19, pendekatan sosioterminologi, kaidah pemadanan, dan keberterimaan istilah Teknik Penyerapan dan Keberterimaan Kata Serapan yang Berhubungan dengan Covid-19: Pendekatan Sosioterminologi Dalam penelitian ini, terdapat dua hal yang dijawab, yaitu kaidah pemadanan istilah apa yang dominan digunakan pada istilah serapan dalam masa pandemi covid-19 dan keberterimaan istilah-istilah tersebut di masyarakat tutur bahasa Indonesia. Metode pengumpulan data yang digunakan adalah metode dokumentasi atau metode simak ditunjang dengan penggunaan kuesioner. Metode analisis data yang digunakan yaitu padan intralingual dan analisis deskriptif. Hasil penelitian menunjukkan terdapat 33 istilah asing yang dipadankan menggunakan teknik penerjemahan, 10 istilah dengan teknik penyerapan, dan 2 istilah dengan teknik gabungan penerjemahan dan penyerapan. Selanjutnya, tingkat keberterimaan istilah serapan dalam masa pandemi virus korona adalah sebanyak 39%, sedangkan 61% sisanya menunjukkan tingkat ketidakberterimaan terhadap istilah serapan dalam masa pandemi virus korona. 1 Pendahuluan Bahasa merupakan salah satu aspek fundamental dalam kehidupan manusia. Segala tindakan manusia dalam kehidupannya selalu bertalian erat dengan bahasa. Dalam hal relasi bahasa dengan masyarakat, kedua hal ini menunjukkan wujud hubungan yang bersifat resiprokal. Artinya, pada kondisi tertentu, perkembangan masyarakat dapat menghendaki perkembangan bahasa, dan di sisi lain, perkembangan bahasa, dapat juga mempengaruhi perkembangan masyarakat. Oleh karena Corresponding author: 1 lmhelmyakbar@gmail.com Copyright © 2021 Lalu Muhammad Helmi, Mahsun, Burhanuddin 139 Helmy, L. M., Mahsun, M & Burhanuddin, B., (2021). Techniques for Absorption into Indonesian and the Acceptance of Loan Words Related to Covid-19: Socioterminology Approach. LITE: Jurnal Bahasa, Sastra, dan Budaya 17 (2), 139-149. https://doi.org/10.33633/lite.v17i2.4761 keadaan masyarakat yang tidak statis, bahasa takluk atas hukum perubahan itu. Salah satu bukti perubahan bahasa itu tampak pada aspek peristilahan. Menurut Kridalaksana (2008), istilah merupakan kata atau gabungan kata yang dengan cermat mengungkapkan konsep, proses, keadaan, atau sifat yang khas dalam bidang tertentu. Pada masa pandemi Corona Virus Disease 2019 (Covid-19) terlihat bahwa bahasa mengalami perubahan. Fakta lingual ini dapat teridentifikasi melalui munculnya topik diskusi ruang publik berkisar ihwal penyakit virus korona berserta istilah-istilah yang mengikutinya seperti social distancing, corona suspect, dan hand sanitizer secara serentak. Istilah-istilah asing tersebut kemudian dibuatkan padanannya dalam bahasa Indonesia. Jika dicermati dari konsep pemadanan istilah yang ada, sistem pemadanan istilah asing pada masa pandemi virus korona berbeda satu dengan yang lain. Sebagai contoh, istilah rapid test yang dipadankan dengan istilah uji cepat. Istilah ini menggunakan teknik pemadanan dengan penerjemahan. Data lain menunjukkan kenyataan yang berbeda, sebagai contoh istilah antiseptic yang dipadankan dengan istilah antiseptik. Istilah ini menggunakan teknik pemadanan dengan penyerapan. Selain itu, bentuk padanan istilah asing yang telah dibakukan itupun belum tentu digunakan oleh penutur bahasa Indonesia. Fakta menunjukkan bahwa istilah asing masih lebih sering digunakan dibandingkan dengan padanan istilah yang telah dibentuk, sebagai contoh istilah Corona virus yang dipadankan menjadi virus korona. Sejak periode Maret 2020 sampai November 2020, data awal menunjukkan bahwa istilah corona virus digunakan sebanyak 3898 kali, sedangkan istilah virus korona sebanyak 205 kali. Faktor lain yang menyebabkan fenomena tersebut adalah terdapat bentukan istilah lain selain yang telah yang telah dipadankan, sebagai contoh istilah asing new normal yang dipadankan menjadi istilah kenormalan baru, tetapi istilah yang lazim digunakan di masyarakat adalah new normal atau normal baru. Penelitian yang mempunyai relevansi dengan penelitian ini di antaranya Qodratillah (2004); Astuti (2007); Darnis (2012); Zaim (2015); Arifin (2018); Sirulhaq dkk. (2018); Darheni (2019); dan Wardani, Vera, dan Nuraiza (2019). Penelitian yang dilakukan Qodratillah (2004) bertujuan mengidentifikasi keseragaman pemakaian istilah di kalangan dokter, baik dengan orang seprofesi maupun dengan orang tak seprofesi serta keseragaman pemakaian istilah di kalangan profesional keuangan dengan orang seprofesi. Penelitian Astuti (2007) bermaksud mengetahui sikap dan penerimaan mahasiswa dan dosen terhadap istilah-istilah bahasa Indonesia, baik itu terjemahan maupun serapan. Penelitian Darnis (2012) merupakan tesis yang bertujuan mengulas hubungan antara kebermanfaatan kaidah pembentukan istilah, yaitu Pedoman Umum Pembentukan Istilah (PUPI) dengan keberterimaan istilah. Selanjutnya, penelitian yang dilakukan oleh Zaim (2015) bertujuan mengungkapkan bagaimana penutur Indonesia menyerap kata asing dalam komunikasi bahasa Indonesia, dan sejauh mana kata serapan asing tersebut berterima dan bermanfaat bagi penutur Indonesia. Penelitian Arifin (2018) bertujuan mendeskripsikan keterbacaan terjemahan dan unsur serapan istilah-istilah teknologi informasi dari bahasa Inggris ke Indonesia. Penelitian yang dilakukan oleh Sirulhaq dkk. (2018) berusaha mengidentifikasi dan mengelaborasi kelemahan KBBI dilihat dari belum adanya bentuk potensial yang ada. Dengan kata lain, penelitian ini menekankan pentingnya bentuk yang potensial untuk dicantumkan sebagai kosakata bahasa Indonesia, tidak hanya mengadopsi kata-kata dari bahasa asing. Penelitian yang dilakukan oleh Darheni (2019) berhasil mengidentifikasi teknik penyerapan istilah asing dalam bidang otomotif dari aspek morfologis dan fonologis. Ditinjau dari segi morfologis, istilah asing dalam bidang otomotif dapat dibentuk melalui proses morfologis, yakni afiksasi, reduplikasi, dan abreviasi. Terakhir, penelitian yang dilakukan oleh Wardani, Vera, Copyright © 2021 Lalu Muhammad Helmi, Mahsun, Burhanuddin 140 Helmy, L. M., Mahsun, M & Burhanuddin, B., (2021). Techniques for Absorption into Indonesian and the Acceptance of Loan Words Related to Covid-19: Socioterminology Approach. LITE: Jurnal Bahasa, Sastra, dan Budaya 17 (2), 139-149. https://doi.org/10.33633/lite.v17i2.4761 dan Nuraiza (2019) bertujuan mendeskripsikan proses pemakaian kata serapan dan istilah asing dalam opini pada surat kabar harian Serambi Indonesia. Penelitian yang memiliki homogenitas dengan penelitian ini memiliki kesamaan dalam aspek objek kajian yakni terminologi atau peristilahan. Perbedaan terdapat pada aspek subjek penelitian, populasi penelitian, dan sampel penelitian. Selain itu, penelitian relevan yang dipaparkan di atas hanya sampai kepada upaya mengidentifikasi teknik pemadanan istilah asing, namun tidak mengkaji kaitan dengan keberterimaan padanan istilah tersebut di masyarakat. Oleh karena itu, penelitian ini menjadi perlu dilakukan guna melengkapi dan menambah khazanah ilmu pengetahuan terkait bidang peristilahan. Mencermati pokok masalah yang akan diuraikan dalam penelitian ini, penulis menggunakan beberapa teori yang akan dijelaskan pada bagian ini yaitu hakikat istilah serapan, penulisan unsur serapan, pedoman umum pembentukan istilah, dan pemadanan istilah. Berikut akan dijelaskan secara berurutan teori-teori tersebut. Kata serapan adalah kata yang diserap dari bahasa lain berdasarkan kaidah bahasa penerima (kbbi.kemdikbud.go.id). Selanjutnya, Kridalaksana (2008), yang kemudian menamakan fenomena ini dengan kata pinjam atau borrowing, menyebutkan bahwa pinjaman merupakan pemasukan unsur fonologis, gramatikal, atau leksikal dalam bahasa atau dialek dari bahasa atau dialek lain karena kontak atau peniruan. Dalam perkembangannya, bahasa Indonesia menyerap unsur dari berbagai bahasa, baik dari bahasa daerah seperti bahasa Jawa, bahasa Sunda, dan bahasa Bali, maupun dari bahasa asing, seperti bahasa Sansekerta, Arab, Portugis, Belanda, Cina, dan Inggris. Berdasarkan taraf integrasinya, unsur serapan dalam bahasa Indonesia dibagi ke dalam dua kelompok besar. Pertama, unsur asing yang belum sepenuhnya terserap ke dalam bahasa Indonesia. Kedua, unsur asing yang penulisan dan pengucapannya disesuaikan dengan kaidah bahasa Indonesia. Dalam hal ini, penyerapan diusahakan agar ejannya diubah seperlunya sehingga bentuk Indonesianya masih dapat dibandingkan dengan bentuk asalnya. Menurut Mulyanto (2017), istilah adalah kata atau frasa yang dipakai sebagai nama atau lambang yang dengan cermat mengungkapkan makna, konsep, proses, keadaan, atau sifat khas dalam bidang ilmu pengetahuan, teknologi, dan seni. Selain itu, tata istilah atau terminologi yang termaktub dalam Mulyanto (2017) didefinisikan sebagai perangkat asas dan ketentuan pembentukan istilah serta kumpulan istilah yang dihasilkannya, sebagai contoh ‘anabolisme’, ‘demokrasi’, ‘pasar modal’, ‘pemerataan’, dan ‘perang elektron’. Pemadanan istilah asing ke dalam bahasa Indonesia (dan jika perlu ke dalam salah satu bahasa serumpun) dilakukan lewat penerjemahan, penyerapan, serta gabungan penerjemahan dan penyerapan. Demi keseragaman, sumber rujukan yang diutamakan adalah bahasa Inggris yang pemakaiannya bersifat internasional karena sudah lazim digunakan oleh para ahli di bidangnya. Penulisan istilah serapan itu dilakukan dengan atau tanpa penyesuaian ejaan berdasarkan kaidah fonotaktik, yakni hubungan urutan bunyi yang diizinkan dalam bahasa Indonesia. 2 Metode Penelitian ini menggunakan pendekatan sosioterminologi. Menurut Tammerman (dalam Darnis, 2012), pendekatan sosioterminologi merupakan kajian tentang terminologi yang berkaitan dengan adat penggunaannya dalam masyarakat. Dalam penelitian ini, yang dimaksud dengan adat penggunaan terminologi adalah terminologi-terminologi serapan yang digunakan dalam masa pandemi virus korona. Jika dikaitkan dengan aspek pengkajiannya, data dalam penelitian ini adalah istilah-istilah yang muncul dan berkaitan dengan dunia kesehatan di masa pandemi virus korona. Oleh karena itu, data Copyright © 2021 Lalu Muhammad Helmi, Mahsun, Burhanuddin 141 Helmy, L. M., Mahsun, M & Burhanuddin, B., (2021). Techniques for Absorption into Indonesian and the Acceptance of Loan Words Related to Covid-19: Socioterminology Approach. LITE: Jurnal Bahasa, Sastra, dan Budaya 17 (2), 139-149. https://doi.org/10.33633/lite.v17i2.4761 yang dimaksud dalam penelitian ini mengacu pada semua tuturan yang mengandung istilah-istilah di masa pandemi. Selanjutnya, dalam rangka mengumpulkan data yang dimaksud di atas, ditempuh tiga tahapan strategis ihwal metode pengumpulan data, yaitu dokumentasi atau metode simak ditunjang dengan penggunaan kuesioner. Metode simak digunakan untuk menyimak keberterimaan dan kebermanfaatan padanan istilah serapan pada masa pandemi virus korona yang digunakan oleh sampel penelitian (responden) dalam bentuk bahasa tulis. Hal ini sesuai dengan pendapat Mahsun (2017) bahwa metode simak atau istilah menyimak tidak hanya berkaitan dengan penggunaan bahasa secara lisan, tetapi juga penggunaan bahasa secara tertulis. Selanjutnya, guna memaksimalkan tahapan pengumpulan data, penelitian juga ditunjang dengan penyebaran kuesioner. Menurut Mahsun (2017), kuesioner merupakan metode pengumpulan data yang digunakan untuk memperoleh data yang lebih terstruktur dan terperinci dari sejumlah besar informan yang dipandang representatif mewakili populasi penelitian. Oleh karena itu, kuesioner tersebut mengandung sejumlah pertanyaan. Adapun metode analisis data yang digunakan yaitu metode padan intraligual seperti yang disarankan Mahsun (2017), serta metode agih (Sudaryanto, 2018), dan metode deskriptif analisis. Di tahap analisis data, metode padan intralingual digunakan untuk menganalisis dan mengelompokkan teknik pemadanan istilah yang digunakan pada istilah-istilah asing di masa pandemi virus korona. Selanjutnya, metode analisis data yang digunakan adalah analisis deskriptif. Menurut Sugiyono, (2018) analisis deskriptif adalah suatu metode yang berfungsi untuk mendeskripsikan atau memberi gambaran terhadap objek yang diteliti melalui data atau sampel yang telah terkumpul sebagaimana adanya tanpa melakukan analisis dan membuat kesimpulan yang berlaku untuk umum atau generalisasi. Dalam penelitian ini, metode analisis deskriptif digunakan untuk menganalisis kuesioner yang telah diberikan kepada responden. Kuesioner yang berisi data balikan responden berkaitan dengan keberterimaan dan kebermanfaatan istilah serapan pada masa pandemi virus korona selanjutnya diolah, dianalisis, dan dijabarkan dalam bentuk tabel. Metode penyajian hasil analisis data yang digunakan dalam penelitian ini adalah metode formal dan informal. Secara sederhana, menurut Mahsun (2017), Sudaryanto (2018), dan Muhammad (2011), metode formal adalah perumusan dengan menggunakan tanda-tanda atau lambang-lambang, sedangkan metode informal adalah perumusan dengan menggunakan kata-kata biasa, termasuk penggunaan terminologi yang bersifat teknis. 3 Hasil dan Pembahasan Terdapat tiga teknik pemadanan istilah asing yang dapat digunakan yaitu, penerjemahan (langsung dan dengan perekaan), penyerapan (penyerapan dengan penyesuaian ejaan dan lafal, penyerapan dengan penyesuaian ejaan tanpa penyesuaian lafal, dan penyerapan tanpa penyesuaian ejaan sekaligus tanpa penyesuaian lafal), dan gabungan penerjemahan dan penyerapan. Dari hasil analisis data, penulis menemukan kelompok data pemadanan istilah serapan dalam masa pandemi virus korona. 1. Kelompok data (I) kontaminasi silang (cross contamination), percikan (droplet), pelindung muka (face shield), pelandaian kurva (flattening the curve), penyanitasi tangan (hand sanitizer), alat pelindung wajah (hazmat mask), Alat Pelindung Diri—APD (hazmat suit), kekebalan kelompok (herd immunity), kasus impor (imported case), penularan lokal (local transmission), karantina wilayah (lockdown), masker (mask), tes serentak (massive test), pandemi (pandemic), protokol (protocol), kenormalan baru (new normal), penjarakan fisik (physical distancing), uji cepat (rapid test), penyaringan (screening), isolasi mandiri (self isolation), karantina mandiri (self quarantine), penjarakan sosial (social distancing), penjarakan media sosial Copyright © 2021 Lalu Muhammad Helmi, Mahsun, Burhanuddin 142 Helmy, L. M., Mahsun, M & Burhanuddin, B., (2021). Techniques for Absorption into Indonesian and the Acceptance of Loan Words Related to Covid-19: Socioterminology Approach. LITE: Jurnal Bahasa, Sastra, dan Budaya 17 (2), 139-149. https://doi.org/10.33633/lite.v17i2.4761 (social media distancing), pembatasan sosial (social restriction), penyintas (survivor), uji usap (tes swab), pistol termo (thermo gun), tes usap tenggorokan (throat swab test), penelusuran; pelacakan (tracing), spesimen (specimen), ventilator (ventilator), Kerja dari Rumah—KDR (Work from Home), Kerja dari Kantor (Work from Office) 2. Kelompok data (II) antiseptik (antiseptic), klorokuin (chloroquine), suspek korona (corona supect), dekontaminasi (decontamination), disinfektan (disinfectant), inkubasi (incubation), isolasi (isolation), respirator (respirator), zoonosis (zoonosis) 3. Kelompok data (III) penyakit koronavirus (coronavirus disease), uji strep cepat (rapid strep test) Berdasarkan hasil analisis terhadap ketiga jenis data di atas, dapat dikatakan bahwa kelompok data (I) termasuk kelompok istilah yang dipadankan menggunakan kaidah penerjemahan, data (II) termasuk kelompok istilah yang dipadankan menggunakan kaidah penyerapan, dan data (III) termasuk kelompok istilah yang dipadankan menggunakan kaidan gabungan penerjemahan dan penyerapan. 3.1 Kaidah Pemadanan Teknik Penerjemahan Dalam kelompok data I, data (1) berupa istilah asing cross contamination yang dipadankan dengan istilah kontaminasi silang. Pemadanan istilah ini menggunakan teknik penerjemahan yakni penerjemahan langsung. Proses penerjemahan tampak pada kata cross yang di dalam bahasa Inggris berarti silang dan kata contamination yang berarti kontaminasi. Data (2) istilah asing droplet dipadankan dengan istilah percikan. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Teknik penerjemahan tampak pada kata droplet yang dalam bahasa Inggris berarti tetesan kecil kemudian dipadankan dengan kata percikan. Data (3) adalah istilah asing face shield dipadankan dengan istilah pelindung muka. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini tampak pada kata face yang di dalam bahasa Inggris berarti muka dan shield yang di dalam bahasa Inggris berarti pelindung. Data (4) istilah asing flattening the curve dipadankan dengan istilah pelandaian kurva. Istilah ini dipadankan menggunakan teknik penerjemahan langsung. Hal ini tampak pada kata flattening yang di dalam bahasa Inggris berarti pelandaian dan curve yang berarti kurva. Data (5) yakni istilah asing hand sanitizier yang dipadankan dengan istilah penyanitasi tangan. Istilah ini dipadankan dengan menggunakan teknik penerjemahan langsung. Hal ini terlihat pada kata hand yang di dalam bahasa Indonesia berarti tangan dan sanitizier yang di dalam bahasa Indonesia berarti penyanitasi. Data (6) adalah istilah asing hazmat mask yang dipadankan dengan istilah alat pelindung wajah. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini dapat dibuktikan pada kata hazmat yang berarti perlengkapan pelindung pribadi dan kata mask yang berarti masker. Gabungan kedua kata tersebut kemudian membentuk istilah hazmat mask yang dipadankan dengan istilah alat pelindung wajah. Data (7) adalah istilah asing hazmat suit yang dipadankan dengan istilah alat pelindung diri. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini dapat dibuktikan pada kata hazmat yang berarti perlengkapan pelindung pribadi dan kata suit yang berarti diri. Data (8) istilah asing herd immunity dipadankan dengan istilah kekebalan kelompok. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini tampak pada kata herd yang di dalam bahasa Indonesia berarti kawanan atau kelompok dan immunity yang berarti kekebalan. Penulis menjumpai data (9) yaitu istilah asing imported case yang dipadankan dengan istilah kasus impor. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini Copyright © 2021 Lalu Muhammad Helmi, Mahsun, Burhanuddin 143 Helmy, L. M., Mahsun, M & Burhanuddin, B., (2021). Techniques for Absorption into Indonesian and the Acceptance of Loan Words Related to Covid-19: Socioterminology Approach. LITE: Jurnal Bahasa, Sastra, dan Budaya 17 (2), 139-149. https://doi.org/10.33633/lite.v17i2.4761 terlihat pada kata imported yang berarti impor dan case yang berarti kasus. Data (10) istilah asing local transmission dipadankan dengan istilah transmisi lokal. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini terlihat pada kata transmission yang berarti penularan dan kata local yang berarti lokal. Data (11) adalah istilah asing lockdown yang dipadankan dengan istilah karantina wilayah. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini terlihat pada istilah lockdown yang jika diterjemahkan langsung berarti kuncitara yang serupa dengan istilah karantina wilayah. Data (12) adalah mask yang dipadankan dengan istilah masker. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini terlihat pada kata mask yang di dalam bahasa Indonesia berarti masker. Data (13) adalah istilah asing massive test yang dipadankan dengan istilah tes serentak. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini terlihat pada kata test yang di dalam bahasa Indonesia berarti tes dan massive yang jika diterjemahkan ke dalam bahasa Indonesia dapat berarti serentak dan besar-besaran. Data (14) adalah istilah asing pandemic yang dipadankan dengan istilah pandemi. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini terlihat pada kata pandemic yang jika diterjemahkan ke dalam bahasa Indonesia berarti pandemi. Data (15) berupa istilah asing protocol yang dipadankan dengan istilah protokol. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini terlihat pada kata protocol yang jika diterjemahkan ke dalam bahasa Indonesia berarti protokol. Data (16) adalah frasa new normal yang dipadankan dengan istilah kenormalan baru. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini terlihat pada kata new yang jika diterjemahkan ke dalam bahasa Indonesia menjadi baru dan normal yang di dalam konteks istilah ini dalam bahasa Indonesia diterjemahkan dengan istilah kenormalan. Data (17) adalah istilah asing physical distancing yang dipadankan dengan istilah penjarakan fisik. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini tampak pada kata physical yang apabila diterjemahkan ke dalam bahasa Indonesia berarti fisik dan distancing yang berarti penjarakan. Data (18) adalah istilah asing rapid test yang dipadankan dengan istilah uji cepat. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini tampak pada kata rapid yang jika diterjemahkan ke dalam bahasa Indonesia berarti cepat dan kata test yang jika diterjemahkan ke dalam bahasa Indonesia berarti uji. Data (19) adalah istilah asing screening yang dipadankan dengan istilah penyaringan. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini dapat dibuktikan jika istilah asing screening diterjemahkan ke dalam bahasa Indonesia menjadi istilah penyaringan. Di data (20), istilah asing self isolation dipadankan dengan istilah isolasi mandiri. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini dapat dibuktikan pada kata self yang jika diterjemahkan ke dalam bahasa Indonesia berarti diri atau mandiri dan kata isolation yang diterjemahkan ke dalam bahasa Indonesia berarti isolasi. Di data (21), penulis menjumpai istilah asing self quarantine yang dipadankan dengan istilah karantina mandiri. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini dapat dibuktikan pada kata self yang jika diterjemahkan ke dalam bahasa Indonesia berarti diri atau mandiri dan kata quarantine yang diterjemahkan ke dalam bahasa Indonesia berarti karantina. Data (22) adalah istilah asing social distancing yang dipadankan dengan istilah penjarakan sosial. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini tampak pada kata social yang apabila diterjemahkan ke dalam bahasa Indonesia berarti sosial dan distancing berarti penjarakan. Copyright © 2021 Lalu Muhammad Helmi, Mahsun, Burhanuddin 144 Helmy, L. M., Mahsun, M & Burhanuddin, B., (2021). Techniques for Absorption into Indonesian and the Acceptance of Loan Words Related to Covid-19: Socioterminology Approach. LITE: Jurnal Bahasa, Sastra, dan Budaya 17 (2), 139-149. https://doi.org/10.33633/lite.v17i2.4761 Data (23) yang merupakan istilah asing social media distancing dipadankan dengan istilah penjarakan media sosial. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini tampak pada kata social yang apabila diterjemahkan ke dalam bahasa Indonesia berarti sosial, kata media berarti media, dan distancing yang berarti penjarakan. Di data (24), penulis menjumpai istilah asing istilah asing social restriction yang dipadankan dengan istilah pembatasan sosial. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini tampak pada kata social yang apabila diterjemahkan ke dalam bahasa Indonesia berarti sosial dan restriction berarti pembatasan atau larangan. Data (25) adalah istilah asing survivor yang dipadankan dengan istilah penyintas. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini dapat dibuktikan bahwa istilah asing survivor jika diterjemahkan ke dalam bahasa Indonesia menjadi penyintas. Data (26) adalah istilah asing swab test yang dipadankan dengan istilah uji usap. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini tampak pada kata swab yang jika diterjemahkan ke dalam bahasa Indonesia berarti usap dan kata test yang jika diterjemahkan ke dalam bahasa Indonesia berarti uji. Di data (27), penulis menjumpai istilah asing thermo gun yang dipadankan dengan istilah pistol termo. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini tampak pada kata gun yang jika diterjemahkan ke dalam bahasa Indonesia berarti pistol dan kata thermo yang jika diterjemahkan ke dalam bahasa Indonesia berarti termo. Data (28) ada istilah asing throat swab test yang dipadankan dengan istilah uji usap tenggorokan. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini tampak pada kata swab yang jika diterjemahkan ke dalam bahasa Indonesia berarti usap, throat yang berarti tenggorokan, dan kata test yang jika diterjemahkan ke dalam bahasa Indonesia berarti uji. Data (29) adalah istilah asing tracing yang dipadankan dengan istilah penelusuran. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini dapat dibuktikan jika istilah asing tracing diterjemahkan ke dalam bahasa Indonesia menjadi istilah penelusuran. Di data (30), ada istilah asing specimen yang dipadankan dengan istilah spesimen. Proses pemadanan istilah ini menggunakan teknik penerjemahan yakni penerjemahan langsung. Hal ini dapat dibuktikan bahwa istilah asing specimen jika diterjemahkan ke dalam bahasa Indonesia menjadi spesimen. Dalam data (31), istilah asing ventilator dipadankan dengan istilah ventilator. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini dapat dibuktikan jika ventilator diterjemahkan ke dalam bahasa Indonesia menjadi ventilator. Data (32) adalah istilah asing work from home yang dipadankan dengan istilah bekerja dari rumah. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini tampak pada kata work yang jika diterjemahkan ke dalam bahasa Indonesia berarti bekerja, from yang berarti dari, dan kata home yang berarti rumah. Di data (33), istilah asing work from office dipadankan dengan istilah bekerja dari rumah. Proses pemadanan istilah ini menggunakan teknik penerjemahan langsung. Hal ini dapat dibuktikan pada kata work yang jika diterjemahkan ke dalam bahasa Indonesia berarti bekerja, from yang berarti dari, dan kata office yang berarti kantor. 3.2 Kaidah Pemadanan Teknik Penyerapan Data (1) adalah istilah asing antiseptic yang kemudian dipadankan dengan istilah antiseptik menggunakan proses pemadanan yakni teknik penyerapan. Teknik penyerapan yang digunakan ialah penyerapan dengan penyesuaian ejaan tanpa penyesuaian lafal. Hal ini mengacu kepada kaidah penyerapan apabila istilah asing berupa sufiks –ic diserap ke dalam bahasa Indonesia akan berubah menjadi –ik. Di data (2), chloroquin dipadankan dengan istilah klorokuin. Proses pemadanan istilah ini menggunakan teknik penyerapan yaitu penyerapan dengan penyesuaian ejaan Copyright © 2021 Lalu Muhammad Helmi, Mahsun, Burhanuddin 145 Helmy, L. M., Mahsun, M & Burhanuddin, B., (2021). Techniques for Absorption into Indonesian and the Acceptance of Loan Words Related to Covid-19: Socioterminology Approach. LITE: Jurnal Bahasa, Sastra, dan Budaya 17 (2), 139-149. https://doi.org/10.33633/lite.v17i2.4761 tanpa penyesuaian lafal. Hal ini berpedoman pada kaidah penyerapan apabila cch dan ch di muka a, o, dan konsonan menjadi k. Di data (3), istilah asing Corona suspect dipadankan dengan istilah suspek korona. Proses pemadanan pada istilah ini menggunakan teknik penyerapan yakni penyerapan dengan penyesuaian ejaan tanpa penyesuaian lafal. Hal ini berpedoman pada kaidah penyerapan c di muka a, u, o, dan konsonan menjadi k dan sufiks –ct dalam bahasa asing diserap menjadi k. Di data (4), istilah asing Coronavirus dipadankan dengan istilah virus korona. Proses pemadanan istilah ini menggunakan teknik penyerapan yakni penyerapan dengan penyesuaian ejaan tanpa penyesuaian lafal. Hal ini mengacu pada kaidah penyerapan c di muka a, u, o, dan konsonan menjadi k. Data (5) adalah istilah asing decontamination dipadankan dengan istilah dekontaminasi. Proses pemadanan istilah ini menggunakan teknik penyerapan yakni penyerapan dengan penyesuaian ejaan dan penyesuaian lafal. Hal ini mengacu pada kaidah penyerapan apabila cc di muka o, u, dan konsonan menjadi k serta sufiks –ion menjadi –asi dalam bahasa Indonesia. Data (6) adalah disinfectant dipadankan menjadi disinfektan. Proses pemadanan istilah ini menggunakan teknik penyerapan yaknik penyerapan dengan penyesuaian ejaan tanpa penyesuaian lafal. Proses penyerapan ini berpedoman pada kaidah ct di muka menjadi konsonan k dan sufiks –ant berubah menjadi –an dalam bahasa Indionesia. Di data (7), penulis menjumpai istilah asing incubation yang dipadankan dengan istilah inkubasi. Proses pemadanan istilah ini menggunakan teknik penyerapan yakni penyerapan dengan penyesuaian ejaan dan penyesuaian lafal. Hal ini seturut dengan kaidah penyerapan sufiks –ion menjadi –asi dalam bahasa Indonesia. Kaidah tersebut juga berimplikasi terhadap penyesuaian lafal. Data (8) istilah asing isolation yang dipadankan dengan istilah isolasi. Proses pemadanan istilah ini menggunakan teknik penyerapan yakni penyerapan dengan penyesuaian ejaan dan penyesuaian lafal. Hal ini seturut dengan kaidah penyerapan apabila sufiks –ion menjadi –asi dalam bahasa Indonesia. Kaidah tersebut juga berimplikasi terhadap penyesuaian lafal. Di data (9), istilah asing respirator dipadankan dengan istilah respirator. Proses pemadanan istilah ini menggunakan teknik penyerapan yakni penyerapan tanpa penyesuaian ejaan dan lafal. Istilah asing respirator diserap secara utuh tanpa pengubahan ejaan dan lafal. Di data (10), istilah asing zoonosis dipadankan dengan istilah zoonosis. Proses pemadanan istilah ini menggunakan teknik penyerapan yakni penyerapan tanpa penyesuaian ejaan dan lafal. Istilah asing zoonosis diserap secara utuh tanpa pengubahan ejaan dan lafal. 3.3 Kaidah Pemadanan Teknik Gabungan Penerjemahan dan Penyerapan Data (1) Coronavirus disease dipadankan dengan istilah penyakit koronavirus. Proses pemadanan istilah ini menggunakan teknik gabungan penerjemahan dan penyerapan. Penyerapan tampak pada pada kata Coronavirus yang dipandakan dengan istilah Koronavirus yang dipadankan berdasarkan kaidah penyerapan apabila c di muka a, u, o, dan konsonan menjadi k. Selanjutnya, penerjemahan dilakukan pada kata disease yang diterjemahkan menjadi kata penyakit. Di data (2), penulis menjumpai istilah asing rapid strep test yang dipadankan dengan istilah uji strep cepat. Proses pemadanan istilah ini menggunakan teknik gabungan penerjemahan dan penyerapan. Teknik penyerapan tampak pada kata strep yang diserap ke dalam bahasa Indonesia menjadi kata strep. Selanjutnya, teknik penerjemahan tampak pada kata rapid dan test yang jika diterjemahkan ke dalam bahasa Indonesia berarti uji dan cepat. Untuk menunjukkan tingkat keberterimaan dari masing-masing istilah serapan pada masa pandemi Covid-19, penulis menampilkan Tabel 1 yang berisi analisis dari kuesioner yang dibagikan ke responden. Copyright © 2021 Lalu Muhammad Helmi, Mahsun, Burhanuddin 146 Helmy, L. M., Mahsun, M & Burhanuddin, B., (2021). Techniques for Absorption into Indonesian and the Acceptance of Loan Words Related to Covid-19: Socioterminology Approach. LITE: Jurnal Bahasa, Sastra, dan Budaya 17 (2), 139-149. https://doi.org/10.33633/lite.v17i2.4761 Tabel 1: Keberterimaan Istilah Serapan dalam Masa Pandemi Virus Korona Istilah Asing Padanan Istilah Antiseptict Chloroquine Corona Suspect Corona Virus Coronavirus Disease Cross Contamination Decontamination Disinfectant Droplet Face Shield Flattening the Curve Hand Sanitizer Hazmat Mask Hazmat Suit Herd Immunity Imported Case Incubation Isloation Local Transmission Lockdown Mask Massive Test New Normal Pandemic Physical Distancing Protocol Rapid Strep Test Rapid Test Respirator Screening Self Isolation Self Quarantine Social Distacing Social Media Distancing Social Restriction Specimen Survivor Swab Test Thermo Gun Throat Swab Test Tracing Ventilator Work From Home Work From Office Zoonois Antiseptik Klorokuin Suspek Korona Virus Korona Penyakit Korona virus Kontaminasi Silang Dekontaminasi Disinfektan Percikan Pelindung Muka Pelandaian Kurva Penyanitasi Tangan Alat Pelindung Wajah Alat Pelindung Diri Kekebalan Kelompok Kasus Impor Inkubasi Isolasi Penularan Lokal Karantina Wilayah Masker Tes Serentak Kenormalan Baru Pandemi Penjarakan Fisik Protokol Uji Strep Cepat Uji Cepat Respirator Penyaringan Isolasi Mandiri Karantina Mandiri Penjarakan Sosial Penjarakan Media Sosial Pembatasan Sosial Spesimen Penyintas Uji Usap Pistol Termo Tes Usap Tenggorokan Penelusuran; Pelacakan Ventilator Kerja Dari Rumah Kerja Dari Kantor Zoonosis Jumlah Rata-rata f 93 28 38 66 22 Pilihan Jawaban Berterima Tidak Berterima (%) f (%) 93% 7 7% 28% 32 32% 38% 62 62% 66% 36 36% 22% 78 78% 17 41 72 5 23 5 6 10 28 8 38 87 90 39 5 97 11 10 93 15 88 5 13 71 2 90 83 12 2 17% 41% 72% 5% 23% 5% 6% 10% 28% 8% 38% 87% 90% 39% 5% 97% 11% 10% 93% 15% 88% 5% 13% 71% 2% 90% 83% 12% 2% 83 59 28 95 77 95 94 90 72 92 62 13 10 61 95 3 89 90 7 85 12 95 87 29 98 10 17 88 98 83% 59% 28% 95% 77% 95% 94% 90% 72% 92% 62% 13% 10% 61% 95% 3% 89% 90% 7% 85% 12% 95% 87% 29% 98% 10% 17% 88% 98% 18 58 8 17 0 7 35 18% 58% 8% 17% 0% 7% 35% 82 42 92 83 100 93 65 82% 42% 92% 83% 100% 93% 65% 67 71 70 55 1719 39 67% 71% 70% 55% 33 29 30 45 2781 61 33% 29% 30% 45% Copyright © 2021 Lalu Muhammad Helmi, Mahsun, Burhanuddin 39% 61% 147 Helmy, L. M., Mahsun, M & Burhanuddin, B., (2021). Techniques for Absorption into Indonesian and the Acceptance of Loan Words Related to Covid-19: Socioterminology Approach. LITE: Jurnal Bahasa, Sastra, dan Budaya 17 (2), 139-149. https://doi.org/10.33633/lite.v17i2.4761 Tabel 1 di atas menunjukkan bahwa tingkat keberterimaan istilah serapan dalam masa pandemi virus korona adalah 39%. Sementara, 61% menunjukkan tingkat ketidakberterimaan terhadap istilah serapan dalam masa pandemi virus korona. Istilah serapan yang paling tinggi tingkat keberterimaannya yakni istilah masker dengan persentase keberterimaan 97%. Selanjutnya, istilah yang paling rendah tingkat keberterimaannya yakni istilah pistol termo dengan persentase tingkat ketidakberterimaan 100%. 4 Simpulan Berdasarkan analisis data yang dilakukan terhadap data yang telah dikumpulkan, penulis dapat menyimpulkan beberapa hal, yaitu istilah serapan dalam masa pandemi virus korona dipadankan dengan menggunakan tiga teknik pemadanan istilah, yaitu teknik penerjemahan, teknik penyerapan, serta teknik gabungan penerjemahan dan penyerapan. Terdapat 33 istilah asing yang dipadankan menggunakan teknik penerjemahan yakni penerjemahan langsung. Selanjutnya, 10 istilah asing dipadankan dengan menggunakan teknik penyerapan yang terdiri atas 6 teknik penyerapan dengan penyesuaian ejaan tanpa penyesuaian lafal, 4 teknik penyerapan dengan penyesuaian ejaan dan penyesuaian lafal, 3 teknik penyerapan tanpa penyesuaian ejaan dan penyesuaian lafal. Lalu, terdapat 2 istilah serapan yang dipadankan menggunakan teknik gabungan penerjemahan dan penyerapan. Tingkat keberterimaan istilah serapan dalam masa pandemi virus korona adalah sebanyak 39% dan 61% lainnya menunjukkan tingkat ketidakberterimaan terhadap istilah serapan dalam masa pandemi virus korona. Istilah serapan yang paling tinggi tingkat keberterimaannya yakni istilah masker dengan persentase keberterimaan 97%. Selanjutnya, istilah yang paling rendah tingkat keberterimaannya yakni istilah pistol termo dengan persentase tingkat ketidakberterimaan 100%. Referensi Arifin, Z. (2018). Keterbacaan Terjemahan dan Serapan Istilah-istilah Teknologi Informasi dalam Teks Panduan Komputer HP: Petunjuk Konfiguras. Surakarta: Universitas Muhammadiyah Surakarta. Astuti, W. (2007). Sikap Bahasa Mahasiswa dan Dosen Terhadap Istilah Terjemahan dan Istilah Serapan Bidang Ekonomi Hasil Mabbim. Humaniora, 67-72. Chaer, Abdul. (2012). Linguistik Umum. Jakarta: Rineka Cipta. Darheni, Nani. (2009). Penyerapan Leksikon Asing dalam Bidang Otomotif ke dalam Bahasa Indonesia: Tinjauan Secara Morfologis dan Fonologis. Jurnal Sosioteknologi, 646 – 666. Darnis, Azhari. (2012). Ketermanfaatan Pedoman Umum Pembentukan Istilah (PUPI) dalam Pembentukan Istilah Bahasa Indonesia. Depok: Universitas Indonesia. Kridalaksana, Harimurti. (2008). Kamus Linguistik. PT Gramedia Pustaka: Jakarta. Kridalaksana, Harimurti. (2009). Pembentukan Kata dalam Bahasa Indonesia. Jakarta: PT Gramedia Pustaka Utama. Mahsun. (2017). Metode Penelitian Bahasa: Tahapan, Strategi, Metode, dan Tekniknya. Depok: Rajawali Press. Muhammad. (2011). Penelitian Bahasa: Paradigma Kualitatif. Yogyakarta: Liebe Book Press. Copyright © 2021 Lalu Muhammad Helmi, Mahsun, Burhanuddin 148 Helmy, L. M., Mahsun, M & Burhanuddin, B., (2021). Techniques for Absorption into Indonesian and the Acceptance of Loan Words Related to Covid-19: Socioterminology Approach. LITE: Jurnal Bahasa, Sastra, dan Budaya 17 (2), 139-149. https://doi.org/10.33633/lite.v17i2.4761 Mulyanto, Firman. (2017). Buku Lengkap EBI (Ejaan Bahasa Indonesia). Yogyakarta: Laksana. Pusat Bahasa. (2007). Pedoman Umum Pembentukan Istilah (PUPI). Jakarta: Departemen Pendidikan Nasional. Qodratillah, Meity Taqdir. (2017). Studi Komparatif tentang Keseragaman Istilah di Bidang Kedokteran dan Keuangan dari Sudut Pembakuan. Depok: Universitas Indonesia. Sirulhaq, Ahmad; Sukri; Jafar, Syamsinas; dan Burhanuddin. (2018). Questioning “The Great Indonesian Dictionary” (KBBI). Advances in Social Science, Education and Humanities Research (ASSEHR).Volume: 228. (p. 28). London: Atlantis Press. Sudaryanto. (2008). Metode dan Aneka Teknik Analisis Bahasa. Yogyakarta: Sanata Dharma University Press. Sugiyono. (2019). Metode Penelitian Kuantitatif, Kualitatif, dan R&D. Bandung: Penerbit Alfabeta. Wardani, Vera, dan Nuraiza. (2019). Proses Pemakaian Kata Serapan dan Istilah Asing dalam Opini pada Harian Serambi Indonesia. Jurnal Riset Sains, 57-66. Zaim, M. (2015). Keberterimaan dan Kebermanfaatan Istilah Serapan Kata Asing dalam Bidang Teknologi Informasi oleh Penutur Bahasa Indonesia. Padang: Universitas Negeri Padang. https://kbbi.kemdikbud.go.id/entri/kata%20serapan Copyright © 2021 Lalu Muhammad Helmi, Mahsun, Burhanuddin 149 

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Vol. 38 (3): 432-433; May - June, 2012 Video Section Single Port Transvesical Prostatectomy _______________________________________________ Fabio C. Vicentini, Marcelo Hisano, Tulio S. Agresta, Claudio B. Murta, Joaquim F. A. Claro Centro de Referência da Saúde do Homem, Hospital de Transplantes do Estado de São Paulo, SP, Brazil ABSTRACT _______________________________________________________________________________________ Purpose: To describe a case of a transvesical prostatectomy performed by a single port technique. Patient and Methods: JLS, 64y, diabetic and hypertense, under treatment of LUTS for 8 years with 4mg doxazosin and 5mg finasteride. The IPSS score was 26. The digital rectal exam showed a more than 60g benign prostate. The Body Mass Index was 28.9. The total PSA was 5.4ng/mL and the free/total PSA was 22%. A 12-fragments prostate biopsy showed BPH. The sonography revealed a 106g prostate and the maximum urinary flow was 12 ml/s. The patient was under general anesthesia and was positioned in dorsal decubitus with Trendelemburg. The bladder was filled until that a bexigoma was visible. A 2 cm longitudinal infra-umbelical incision was done. The Gel Point Single Port System (Applied, Ca, USA) was placed inside the bladder and the pneumovesicum was done until 10mmHg. A peri-bladder neck incision was done and the adenoma dissection was performed until its remotion. The hemostasia was done under vision. A 3-way 24-Fr Foley catheter and an 8-Fr plastic catheter were placed inside the bladder. The adenoma was removed and the bladder and the abdominal wall were closed. Results: The procedure took 55 minutes and the blood loss was 180 ml. The patient evolved uneventfully, the bladder irrigation stayed for 24 h, the hemoglobin drop was 2.4g/dL and the patient was discharge after 36 hours. The urethral catheters stayed for 5 days. The postoperative IPSS was 6 and the maximum flow was 26 ml/s. Conclusion: The surgery was safe and effective, showing that the single port transvesical prostatectomy can be an option in the surgical treatment of large prostates. ARTICLE INFO Available at: www.brazjurol.com.br/videos/may_june_2012/Vicentini_432_433video.htm Int Braz J Urol. 2012; 38 (Video #4): 432-433 ________________ ______________________ Correspondence address: Submitted for publication: February 15, 2012 Dr. Fabio C. Vicentini Centro de Referência da Saúde do Homem, Hospital de Transplantes do Estado de Sao Paulo, SP, Brazil Rua Dr. Alceu de Campos Rodrigues, 46 / 21 Sao Paulo, SP, 04544-000, Brazil E-mail: fabio@drfabiovicentini.com.br ________________ Accepted after revision: May 12, 2012 432 IBJU | Video Section Editorial Comment In this video by Vicentini et al., the authors employ a novel single port transvesical approach for the surgical management of benign prostatic hyperplasia (BPH). I applaud the authors on employing such a well suited minimally invasive surgical approach to BPH refractory to current medical therapies. As a host of alternate surgical therapies arise for BPH including Holmium-YAG laser enucleation/vaporization and Greenlight laser vaporization, the ultimate question is which one provides the best outcomes in terms of longterm improved urinary stream, minimal irritative post-treatment voiding symptoms, optimal surgical outcomes (i.e. quickest convalescence, less perioperative pain, decreased blood loss/necessity for blood transfusion, and lowest risk of complications), as well as the ever increasing concern of treatment specific healthcare cost. Clearly, this is a complex question which can’t be answered through a single video presentation or case series but will require multi-arm prospective studies to be conducted. I encourage the urological society at large to consider doing such studies because it is only though such efforts that we can ultimately answer the question of what is the best option. It is likely that certain patients may do better with one specific surgical option versus another based on the size of the gland, specific surgical anatomy e.g. prominent median lobe, and baseline irritative voiding symptoms. Dr. Philippe E. Spiess Assistant Professor Dept of Genitourinary Oncology H. Lee Moffitt Cancer Center Video Section Editor, International Braz J Urol 12902 Magnolia Drive Office 4035C Tampa, Florida USA 33612 Fax: + 1 813 745-8494 E-mail: philippe.spiess@moffitt.org 433 

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RESEARCH ARTICLE The Phylogenetically-Related Pattern Recognition Receptors EFR and XA21 Recruit Similar Immune Signaling Components in Monocots and Dicots Nicholas Holton1, Vladimir Nekrasov1, Pamela C. Ronald2, Cyril Zipfel1* 1 The Sainsbury Laboratory, Norwich Research Park, Norwich, United Kingdom, 2 Department of Plant Pathology and the Genome Center, University of California, Davis, Davis, California, United States of America * cyril.zipfel@tsl.ac.uk Abstract OPEN ACCESS Citation: Holton N, Nekrasov V, Ronald PC, Zipfel C (2015) The Phylogenetically-Related Pattern Recognition Receptors EFR and XA21 Recruit Similar Immune Signaling Components in Monocots and Dicots. PLoS Pathog 11(1): e1004602. doi:10.1371/journal.ppat.1004602 Editor: Wenbo Ma, University of California Riverside, UNITED STATES Received: June 1, 2014 Accepted: December 4, 2014 Published: January 21, 2015 Copyright: © 2015 Holton et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was funded by the Gatsby Charitable Foundation, the Two Blades Foundation and the Citrus Research & Development Foundation to CZ. 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. During plant immunity, surface-localized pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs). The transfer of PRRs between plant species is a promising strategy for engineering broad-spectrum disease resistance. Thus, there is a great interest in understanding the mechanisms of PRR-mediated resistance across different plant species. Two well-characterized plant PRRs are the leucine-rich repeat receptor kinases (LRR-RKs) EFR and XA21 from Arabidopsis thaliana (Arabidopsis) and rice, respectively. Interestingly, despite being evolutionary distant, EFR and XA21 are phylogenetically closely related and are both members of the sub-family XII of LRR-RKs that contains numerous potential PRRs. Here, we compared the ability of these related PRRs to engage immune signaling across the monocots-dicots taxonomic divide. Using chimera between Arabidopsis EFR and rice XA21, we show that the kinase domain of the rice XA21 is functional in triggering elf18-induced signaling and quantitative immunity to the bacteria Pseudomonas syringae pv. tomato (Pto) DC3000 and Agrobacterium tumefaciens in Arabidopsis. Furthermore, the EFR:XA21 chimera associates dynamically in a ligand-dependent manner with known components of the EFR complex. Conversely, EFR associates with Arabidopsis orthologues of rice XA21-interacting proteins, which appear to be involved in EFR-mediated signaling and immunity in Arabidopsis. Our work indicates the overall functional conservation of immune components acting downstream of distinct LRR-RK-type PRRs between monocots and dicots. Author Summary Pests and diseases cause significant agricultural losses. Plants recognize pathogen-derived molecules via plasma membrane-localized immune receptors (called pattern recognition receptors or PRRs), resulting in pathogen resistance. In recent years, the transfer of PRRs PLOS Pathogens | DOI:10.1371/journal.ppat.1004602 January 21, 2015 1 / 22 Conservation of Immune Signaling between Monocot and Dicot PRRs across plant species has emerged as a promising biotechnological approach to improve crop disease resistance. Successful transfers of PRRs suggest that immune signaling components are conserved across plant species. In this study, we demonstrate that the PRR XA21 from the monocot plant rice is functional in the dicot plant Arabidopsis thaliana (Arabidopsis) and that it confers quantitatively enhanced resistance to bacteria. Furthermore, we show that the rice XA21 and the Arabidopsis EFR, which are evolutionary-distant but phylogenetically closely related, recruit similar signaling components for their function, revealing an overall conservation of immune pathways across monocots and dicots. These findings demonstrate evolutionary conservation of downstream signaling from PRRs and indicate that transfer of PRRs is possible between different plant families, but also between monocots and dicots. Introduction The first line of innate immunity in plants is conferred by surface-localized pattern recognition receptors (PRRs) that recognize microbial patterns, referred to as pathogen-associated molecular patterns (PAMPs). PRR-triggered immunity (PTI) is involved in basal resistance to adapted pathogens and in non-host resistance to non-adapted pathogens. Furthermore, PAMP recognition triggers both local and systemic immune responses in plants. Plant PRRs are either receptor kinases (RKs) or receptor-like proteins (RLPs). RKs are modular proteins with an extracellular domain potentially involved in ligand binding, a single pass transmembrane domain and an intracellular kinase domain that relays downstream signaling. In contrast to RKs, RLPs do not possess any kinase domain and only have a short cytoplasmic tail that lacks any obvious signaling motifs. Thus, RLPs most likely form heteromeric complexes with RKs or other cytoplasmic kinases to transduce signaling upon ligand perception. RKs represent one the largest plant protein families, with leucine-rich repeat (LRR)-type RKs being the largest group of RKs [1]. For example, the Arabidopsis and rice genomes encode >600 and >900 RKs of which >220 and >290 are LRR-RKs, respectively [2–5]. Plant RKs are evolutionary related and have mostly evolved through duplication events leading to the expansion of the family [3,5–8]. The first LRR-RK implicated in plant immunity was rice XA21, which confers resistance to the bacterium Xanthomonas oryzae pv. oryzae (Xoo, the causal agent of bacterial blight) [9]. The ligand for XA21 is still unknown [10]. XA21 belongs to the sub-family XII of LRR-RKs (LRR-XII), which is one of the most expanded sub-families of LRR-RKs in rice with >150 members. This family also contains numerous members in other sequence genomes, such as poplar (Populus trichocarpa) with >40 members [11], tomato (Solanum lycopersicum) with >50 members [12] and grapevine (Vitis vinifera) with >34 members [4]. In contrast, Arabidopsis thaliana (hereafter referred to as Arabidopsis) only contains 10 sub-family LRRXII members [8]. Despite its relatively small size, the Arabidopsis LRR-XII sub-family contains FLS2 and EFR that recognize bacterial flagellin (or the derived peptide flg22) and elongation factor TU (EF-Tu) (or the derived peptide elf18), respectively [13,14], and which are amongst the best-characterized PRRs in plants. Notably, XA21, FLS2 and EFR are non-RD kinases (where RD refer to conserved Arg and Asp residues in the kinase subdomain VIb), a feature correlated with a function in innate immunity across kingdoms [15,16]. The presence of nonRD kinase domains in most members of the sub-family LRR-XII in diverse plant species suggests that this sub-family encodes mostly for PRRs or PRR-associated RKs [16]. PLOS Pathogens | DOI:10.1371/journal.ppat.1004602 January 21, 2015 2 / 22 Conservation of Immune Signaling between Monocot and Dicot PRRs The transfer of PRRs between plant species is a promising approach for engineering broadspectrum disease resistance that may be durable [17,18]. Such transfer has already been used in conventional breeding programs to generate hybrids with PRR-mediated resistance from wild relatives as single or pyramided loci [19]. For example, XA21 was originally introgressed from a wild species of rice (Oryza longistaminata) into cultivated rice (Oryza sativa) [9,20]. However, these approaches are restricted by inter-species incompatibilities. In recent years, several reports have described the successful transgenic transfer of PRRs into phylogenetically diverse species leading to improved disease resistance. For example, transgenic expression of Arabidopsis EFR into the distantly related solanaceous dicot species Nicotiana benthamiana and tomato provides broad-spectrum resistance to bacteria belonging to different genera [21]. Conversely, transfer of the tomato LRR-RLP Ve1 into Arabidopsis confers resistance to different Verticillium species [22]. Also, rice XA21 has been introduced into the monocot banana (Musa sp.) and confers resistance to Banana Xanthomonas wilt caused by X. campestris pv. musacearum [23]. Furthermore, transgenic expression of rice XA21 in the dicot species tomato and sweet orange (Citrus x sinensis) results in improved resistance to bacterial wilt caused by Ralstonia solanacearum and citrus canker caused by X. axonopodis pv. citri, respectively [24,25]. Importantly, these examples suggest that PRR-mediated signaling pathways are conserved across plant species, families, and even clades, and that the transferred PRRs can recruit biochemically these potentially conserved immune components. A number of genetic and biochemical studies have identified components regulating PRR biogenesis, accumulation and downstream signaling in Arabidopsis and rice [26–28]. Consistent with their close phylogenetic relationship [2–5], both EFR and XA21 heavily rely on evolutionary conserved endoplasmic reticulum-quality control (ER-QC) components for proper folding and accumulation [29–35]. These requirements may illustrate the relatively younger age of EFR and XA21 in evolutionary terms, since these PRRs seem to have evolved recently in Brassicaceae and in wild rice species, respectively. In contrast, FLS2, which is more distantly related to XA21 than is EFR, is conserved in all vascular plants [36] and does not show similar genetic and biochemical ER-QC requirements for its biogenesis. In terms of downstream signaling, FLS2 and EFR engage largely overlapping components leading to congruent immune outputs, including transcriptional changes [13,37]. In summary, they form ligand-dependent heteromers with the LRR-RK BAK1/SERK3 (and related SERK proteins) that acts as co-receptor and positive regulator [38–41]. Receptor-like cytoplasmic kinases (RLCKs), such as BIK1 and PBL1, are direct substrates of the FLS2/EFR-BAK1 complexes and their ligand-induced phosphorylation lead to activation of specific immune outputs [42–44]. Recently, it has been shown that activated BIK1 directly phosphorylates the NADPH oxidase RBOHD to trigger an apoplastic burst of reactive oxygen species (ROS), one of the hallmark early PTI responses [45,46]. Other immune responses triggered upon flg22 or elf18 perception include activation of mitogen-activated protein (MAP) and calcium-dependent protein kinases (MAPKs and CDPKs), callose deposition and expression of immune-related genes [36]. Prior ligand-binding, the formation of the FLS2(/EFR)-BAK1 is inhibited by the BAK1-interacting LRR-RK BIR2 [47]. Upon heteromerization with BAK1, the BAK1-associated E3 ligases PUB12 and PUB13 are transferred to FLS2 (and potentially EFR) to ubiquitinate the ligand-bound PRR, targeting it to degradation [48]. Also, ligand-bound FLS2 is endocytosed [49]. These two mechanisms are most likely required to enable replenishment of the plasma membrane with ligand-free PRRs. Signaling components acting downstream of XA21 have been mostly identified through interaction studies. In the absence of ligand, XA21 is kept inactive by the ATPase XB24 that triggers XA21 auto-phosphorylation, keeping its kinase inactive [50]. After activation and XB24 dissociation, XA21 activity is inhibited by the protein phosphatase 2C (PP2C) XB15 through PLOS Pathogens | DOI:10.1371/journal.ppat.1004602 January 21, 2015 3 / 22 Conservation of Immune Signaling between Monocot and Dicot PRRs dephosphorylation [51]. The E3 ligase XB3 and the plant-specific ankyrin-repeat (PANK) protein XB25 are XA21-interacting proteins that are required for its accumulation [52,53]. Mechanisms that positively regulate XA21-mediated early immune signaling are mostly undefined. Only very recently, it was shown that the rice SERK protein OsSERK2 constitutively associates with XA21 and positively regulates XA21-mediated immunity to Xoo [54]. Surprisingly, despite belonging to the same sub-family of LRR-RKs, a comparison of the currently known molecular mechanisms underlying immune signaling triggered by FLS2/EFR and XA21 in Arabidopsis and rice, respectively, reveals very little similarity. The lack of a proven ligand for XA21 [10] and the lack of robust assays for XA21-mediated immune signaling have hindered the study of dynamic immune signaling upon XA21 activation. In this study, we directly compared the ability of two evolutionary-distant, yet phylogenetically closely related PRRs to engage immune signaling in a single plant species (Arabidopsis thaliana). Using chimera between Arabidopsis EFR and rice XA21, we show that the kinase domain of the rice XA21 is functional in Arabidopsis to induce signaling and quantitative immunity to the bacterium Pseudomonas syringae pv. tomato (Pto) DC3000 and Agrobacterium tumefaciens. In addition, our results suggest that the XA21 extracellular domain may perceive a ligand derived from Pto DC3000. The EFR:XA21 chimera associates dynamically in a liganddependent manner with known components of the EFR complex. Conversely, EFR associates with Arabidopsis orthologues of rice XA21-interacting proteins, which appear to be involved in EFR-mediated signaling and immunity in Arabiodpsis. Our work indicates the overall functional conservation of immune components acting downstream of distinct LRR-RK-type PRRs between monocots and dicots. Results/Discussion The XA21 intracellular domain confers immune signaling in dicots To study the functionality of XA21 in dicots, we constructed chimeric receptors between EFR and XA21, and compared them to full-length EFR and XA21 proteins. Chimeras (EFR:XA21 and XA21:EFR) were produced by fusion of the extracellular domain with the transmembrane/ intracellular domain, at the external boundary of the transmembrane domain (Fig. 1A). Previously Albert et al. [55] examined the functionality of EFR:FLS2 chimeras and found fusion at an equivalent position was functional. All proteins were C-terminally tagged with green fluorescent protein (GFP) to check protein accumulation. Because the XA21 ligand is currently unknown, the availability of the EFR:XA21 chimera allows assessment of the activation of the XA21 intracellular domain in response to elf18 binding to the EFR extracellular domain. We first tested the functionality of these receptors by expressing them transiently in Nicotiana benthamiana, which lacks endogenous EFR and thus does not perceive elf18 [13,21]. Immunoblot analysis indicated that all proteins accumulated (Fig. 1B) and localized predominantly to the plasma membrane (S1 Fig.). Activation of these receptors was then tested by analyzing the ability to confer an elf18-induced ROS burst. Both EFR and EFR:XA21 expression allowed the similar production of ROS in response to elf18 (Fig. 1C). However, the expression of neither XA21 or XA21:EFR led to production of ROS greater than that seen when the enzyme β-glucuronidase (GUS; used as a transformation control) was expressed (Fig. 1C). We then sought to confirm these results upon stable expression in Arabidopsis. To this end, we generated transgenic lines in a null efr-1 mutant background with the constructs. We selected two independent homozygous lines that properly accumulated the tagged receptors (Fig. 1D) for further analysis. As observed upon transient expression in N. benthamiana, only EFR and EFR:XA21 conferred elf18 responsiveness (Fig. 1E). Notably, the total amount of ROS generated correlated with protein accumulation (Fig. 1D). PLOS Pathogens | DOI:10.1371/journal.ppat.1004602 January 21, 2015 4 / 22 Conservation of Immune Signaling between Monocot and Dicot PRRs Figure 1. The EFR:XA21 chimera confers immune signaling in N. benthamiana and A. thaliana. A. Schematic representation of chimeric PRRs generated in this study. Hatched shading indicates the transmembrane region. Amino acids at the boundary of the protein fusion are indicated. B. Accumulation of GUS, EFR:GFP, EFR:XA21:GFP; XA21:GFP; XA21:EFR:GFP proteins transiently expressed under the control of the 35S promoter in N. benthamiana. Protein was extracted at the same time point after infection as the ROS assays presented in (C) were performed. C. Elf18-induced ROS production following transient expression of GUS, EFR:GFP, EFR:XA21:GFP, XA21:GFP or XA21:EFR:GFP transiently expressed under the control of the 35S promoter in N. benthamiana. ROS production is represented as the total of photons emitted during 40 min after treatment with 100 nM elf18. Values are averages ±SE (n = 12). D. Accumulation of GFP fusions in A. thaliana efr-1 and efr-1 transgenic homozygous lines stably expressing EFR:GFP, EFR:XA21: GFP, XA21:EFR:GFP or XA21:GFP under the control of the 35S promoter. Two independent lines are shown; the same as those used for ROS induction in (E). Western blots were performed with HRP-conjugated anti-GFP antibodies. Membranes were stained with Coomassie brilliant blue (CBB) to document equal protein loading. E. Elf18-induced ROS production in A. thaliana Col-0, efr-1 and efr-1 transgenic homozygous lines stably expressing EFR:GFP, EFR: XA21:GFP, XA21:EFR:GFP or XA21:GFP under the control of the 35S promoter. ROS production is represented as the total of photons emitted during 40 min after treatment with 100 nM elf18. Values are averages ±SE (n = 12). Experiments performed three times with similar results. Asterisks indicate significant differences from efr-1; p < 0.001 (Student’s t-test). doi:10.1371/journal.ppat.1004602.g001 PLOS Pathogens | DOI:10.1371/journal.ppat.1004602 January 21, 2015 5 / 22 Conservation of Immune Signaling between Monocot and Dicot PRRs To further characterize the function of the XA21 cytoplasmic domain, we compared quantitatively the ability of EFR and EFR:XA21 to induce elf18-induced marker gene expression when expressed stably in Arabidopsis. Notably, the expression levels of PR1, FRK1, PHI1 and NHL10 following efl18 treatment was comparable in Col-0, EFR and EFR:XA21 plants (S2 Fig.). Importantly, the basal expression of these immune marker genes was not affected in the transgenic lines expressing EFR or EFR:XA21 (S2 Fig.), indicating that these plants do not show any sign of constitutive immune responses. Together, these data showed that the chimeric receptor EFR:XA21 is capable of responding to elf18, demonstrating that the XA21 cytoplasmic domain can substitute for the EFR cytoplasmic domain to induce immune outputs. However, the XA21 extracellular domain did not confer elf18 responsiveness, indicating that XA21 does not bind elf18. Expression of XA21 and the EFR:XA21 chimera in Arabidopsis increases immunity to pathogenic bacteria Next, we determined if the signaling outputs triggered by the XA21 intracellular domain could lead to increased immunity to pathogens in Arabidopsis. Pre-treatment of Arabidopsis plants with elf18 restricts growth of the plant pathogenic bacterium Pseudomonas syringae pv. tomato (Pto) DC3000 by about 1 log, which is lost in the null efr-1 mutant (Zipfel et al., 2006). The transgenic lines expressing EFR produced the same level of elf18-induced resistance than wildtype Col-0 plants, while the EFR:XA21 lines produced a lower level of elf18-induced resistance (Fig. 2A). This is on contrast to the transgenic lines expressing XA21 and XA21:EFR, which were insensitive to elf18 as observed in the parental efr-1 line (Fig. 2A). Letters indicate statistically significant differences from Tukey’s HSD mean separation (p < 0.05). EFR expression also contributes to resistance to Agrobacterium tumefaciens by recognising the endogenous EF-Tu [13,21], which can be measured in an Agrobacterium-mediated transient expression assay using an intronic GUS transgene followed by a colorimetric or fluorometric assay to quantify GUS enzymatic activity. Leaves of the null efr-1 mutants allow more GUS expression than wild-type Col-0 leaves (Fig. 2B, C), as previously observed [13]. The transgenic lines expressing EFR or EFR:XA21 complemented the efr-1 phenotype and exhibited low GUS expression to a level comparable to Col-0 (Fig. 2B, C). In contrast, levels of GUS expression observed in the XA21 and XA21:EFR lines were similar to those observed in efr-1 (Fig. 2B, C). Thus, the presence of the intracellular domain of XA21 enabled transmission of elf18 or EF-Tu perception by the EFR extracellular domain into an immune output leading to improved pathogen resistance. We were also interested to test the potential contribution of the XA21 intracellular domain in the basal resistance to Pto DC3000. For this, we used the hypo-virulent strains Pto DC3000 COR− after spray-inoculation, an assay that has been used previously to reveal the contribution of FLS2 and EFR to this bacterium [33,56,57]. This strain is affected in biosynthesis of the toxin coronatine, which is a jasmonic acid mimic involved in the suppression of pre- and post-invasive immunities [58]. As previously reported [56], efr-1 plants are more susceptible to Pto DC3000 COR− (Fig. 2D). Consistent with our previous results (Figs. 1 C, E and 2 A–C), expression of EFR or EFR:XA21 was capable of complementing efr-1 (Fig. 2D). Surprisingly, expression of XA21 or XA21:EFR in two independently transformed lines also resulted in improved resistance to Pto DC3000 COR− in the efr-1 background (Fig. 2D). This effect cannot be caused by recognition of the elf18 epitope derived from Pto EF-Tu, as these receptors did not confer elf18-dependent responsiveness in previous assays (Figs. 1 C, E and 2 A–C). These data suggest that the XA21 ectodomain may be able to perceive a ligand derived from Pto DC3000. PLOS Pathogens | DOI:10.1371/journal.ppat.1004602 January 21, 2015 6 / 22 Conservation of Immune Signaling between Monocot and Dicot PRRs Figure 2. Bacterial resistance conferred by EFR, XA21 and chimeric receptors in A. thaliana. A. Elf18-induced resistance in A. thaliana Col-0, efr-1 and efr-1 transgenic homozygous lines stably expressing EFR:GFP, EFR:XA21:GFP, XA21:GFP or XA21:EFR:GFP under the control of the 35S promoter. Plants were pre-treated with mock (water) or 1 μM elf18 for 24 h before infection with Pto DC3000 (OD600 = 0.0002). Bacterial populations were scored at 2 dpi. Values are averages ±SE (n = 4). Infections were repeated four times with similar results. B. Agrobacterium-mediated transient expression of a 35S::GUS transgene in A. thaliana Col-0, efr-1 and efr-1 transgenic homozygous lines stably expressing EFR:GFP, XA21:GFP, EFR:XA21:GFP or XA21:EFR:GFP under the control of the 35S promoter. Leaves were stained for GUS activity at 3 dpi. Leaves shown are representative of the variation in GUS staining observed. The experiment was repeated four times with similar results. C. Fluorimetric quantification of GUS activity. Values are averages ±SE (n = 6). The experiment was repeated three times with similar results. D. Susceptibility of A. thaliana Col-0, efr-1 and efr-1 transgenic homozygous lines stably expressing EFR:GFP, XA21:GFP, EFR:XA21:GFP, or XA21:EFR:GFP under the control of the 35S promoter to Pto DC3000 COR− (OD600 = 0.2; spray inoculation). Bacterial populations were scored at 3 dpi. Values are averages ±SE (n = 4). Different letters indicate significantly different values at p <0.05 (one-way ANOVA). The experiment was repeated 5 times with similar results. doi:10.1371/journal.ppat.1004602.g002 It cannot be excluded that ectopic over-expression of XA21 or XA21:EFR may result in some degree of auto-activation leading to a ligand-independent enhanced disease resistance. To test if these lines show signs of constitutive immune responses, basal PR1 expression (as a marker for SA perception/signaling) and MAPK activity were measured (S3 Fig.). No PLOS Pathogens | DOI:10.1371/journal.ppat.1004602 January 21, 2015 7 / 22 Conservation of Immune Signaling between Monocot and Dicot PRRs significant basal PR1 expression or MAPK activity was observed, suggesting that immune responses are not auto-activated in these lines. Expression of XA21 or XA21:EFR may enable the recognition of bacteria expressing the XA21 ligand in the phyllosphere or rhizosphere that would ‘prime’ immune responses. However, we did not observe any necrosis or growth defects in these lines, and they were also not more resistant to Agrobacterium or Pto DC3000 upon infiltration (Fig. 2A–C), which would be expected if these hypotheses were correct. XA21-mediated resistance in rice relies on the expression of several components genetically identified in X. oryzae pv. oryzae, namely: the predicted type I secretion components raxA, raxB and raxC; raxP, raxQ and raxST, involved in the regulation of 30 -phosphoadenosine 50 -phosphosulfate generation and sulfation; and raxR/H, which controls hrp gene expression [59–61]. Pto DC3000 possesses many of these components, except for raxST [62]. While these components are important for generating a functional XA21 ligand in Xoo, it is not known whether these bacterial genes are required for production of a functional XA21 ligand in other bacterial species. Notably, transgenic XA21 expression in tomato provides resistance to Ralstonia solanacearum [24], which contains a raxST homologue unlike Pto, which lacks raxST These data and the results presented here suggest that XA21 may recognize a ligand present in many bacterial pathogens and that its function is not restricted to recognition of Xanthomonas species. The future identification of the XA21 ligand(s) is required to address these questions. The availability of the Arabidopsis XA21:EFR line generated in this study and of well-characterized quantitative early immune outputs triggered by the EFR intracellular domain may be helpful to tackle the challenge of identifying the elusive XA21 ligand. The EFR:XA21 chimera associates dynamically with BAK1 and BIK1 in a ligand-dependent manner The functionality of the XA21 intracellular domain in conferring immune signaling and immunity upon elf18 perception by the EFR extracellular domain (Figs. 1 and 2) suggests that the EFR:XA21 chimera recruit similar downstream signaling components as EFR. BAK1 associates dynamically with FLS2 and EFR in a ligand-dependent manner [39–41,63]. A recent structural study demonstrated that flg22 acts as ‘molecular glue’ between the flg22bound FLS2 and BAK1 enabling the quasi-instantaneous complex formation between the two proteins [38]. Thus, BAK1 acts as a co-receptor. Notably, a similar mechanism has been observed for the ligand-induced complex formation between the brassinosteroid receptor BRI1 and its co-receptors BAK1 and SERK1 [64,65]. To investigate if the EFR:XA21 chimera could also associated with BAK1 in a ligand-dependent manner, GFP-tagged EFR and EFR:XA21 were transiently co-expressed with hemagglutinin (HA)-tagged BAK1 in N. benthamiana followed by treatment with mock or elf18 for 10 min. Immunoprecipitation of EFR or EFR:XA21 using GFP-Trap beads demonstrated that BAK1 only co-precipitated following elf18 treatment (Fig. 3A,D). In addition, ligand-dependent association of native BAK1 was also observed in transgenic Arabidopsis plants expressing EFR or EFR:XA21 (Fig. 3B). To determine whether the XA21 cytoplasmic domain is required for the interaction with BAK1, we created truncated chimeras that contain only the extracellular and transmembrane domains (including a short positively charged region of the intracellular juxtamembrane). Like the full-length receptor, interaction with BAK1 was observed only following elf18 treatment (S4 Fig.). However this interaction is not sufficient to elicit elf18-induced ROS burst. These data indicate that the extracellular domain is sufficient for BAK1 interaction, as previously shown in the case of the association between FLS2 and BAK1 [38]. Interestingly, a rice BAK1 orthologue, OsSERK2, was very recently shown to associate constitutively with XA21 and OsFLS2 [54], suggesting distinct biochemical mechanisms of PLOS Pathogens | DOI:10.1371/journal.ppat.1004602 January 21, 2015 8 / 22 Conservation of Immune Signaling between Monocot and Dicot PRRs Figure 3. Dynamic association of EFR and the EFR:XA21 chimera with BAK1 and BIK1 in planta. A. Co-immunoprecipitation of transiently-expressed EFR:GFP and EFR:XA21:GFP with BAK1:HA in N. benthamiana. Leaves were treated with 100 nM elf18 (+) or mock treated with water (−) for 10 min. Immunoprecipitation (IP) was performed with GFP-Trap agarose beads. Western blots were performed with HRP-conjugated anti-GFP and anti-HA antibodies. Experiments were performed at least twice with similar results. B. Co-immunoprecipitation with BAK1 in A. thaliana Col-0 and efr-1 transgenic homozygous lines stably expressing EFR:GFP or EFR:XA21:GFP under the control of the 35S promoter. Leaves were treated with 100 nM elf18 (+) or mock treated with water (−) for 10 min. Immunoprecipitation (IP) was performed with GFP-Trap agarose beads. Western blots were performed with HRPconjugated anti-GFP, anti-HA or anti-BAK1 antibodies. Experiments were performed at least twice with similar results. C. Co-immunoprecipitation of transiently-expressed EFR:GFP and EFR:XA21:GFP with BIK1:HA in N. benthamiana. Leaves were treated with 100 nM elf18 (+) or mock treated with water (−) for 10 min. Immunoprecipitation (IP) was performed with GFP-Trap agarose beads. Western blots were performed with HRP-conjugated anti-GFP and anti-HA antibodies. Experiments were performed at least twice with similar results. D, E. Quantitation of data presented in A and C. Values are the average ratio of BAK:HA or BIK:HA to EFR:GFP ±SE (n = 3). Asterisks indicate statistical significance of mock compared to elf18 treated, p < 0.001 (Student’s t-test). ns is not significant at p <0.05. nd is not determined. doi:10.1371/journal.ppat.1004602.g003 association with SERK proteins between monocots and dicots. Importantly, it remains to be tested if the association between XA21-OsSERK2 and OsFLS2-OsSERK2 would be stronger upon ligand treatment. Our data suggest that the constitutive interaction of PRRs with OsSERK2 observed in rice is not due to a property of the XA21 intracellular domain. Such constitutive interaction must therefore be dependent on OsSERK2 or the extracellular domains of XA21 and OsFLS2. It is also possible that components that control the interactions between SERKs and RKs plays distinct functions between monocots and dicots, or that they are absent in monocots. For example, Arabidopsis BIR2 has been recently demonstrated to interact with BAK1 and prevent or reduce its association with FLS2 [47]. While homologues of BIR2 are present in rice, it is not known if they associate with OsSERK2 and control its interaction with PRRs. The RLCKs BIK1 and related PBL proteins are direct substrates of EFR and other PRRs [43,44,66]. Transient co-expression of GFP-tagged EFR or EFR:XA21 with HA-tagged BIK1 was performed to determine the interaction with BIK1. BIK1 constitutively associated with PLOS Pathogens | DOI:10.1371/journal.ppat.1004602 January 21, 2015 9 / 22 Conservation of Immune Signaling between Monocot and Dicot PRRs both EFR and EFR:XA21 (Fig. 3C,D). Following elf18 treatment, we observed partial dissociation of these complexes, similar to that previously reported between EFR and BIK1 in Arabidopsis [43]. These results illustrate that the XA21 intracellular domain can dynamically associate with BIK1. BIK1 homologues exist in rice and other monocots. One of these, OsRLCK185, interacts with the LysM-RK CERK1 involved in chitin perception and is involved in chitin-mediated signaling [67]. Notably, in Arabidopsis, BIK1 is also involved in chitin signaling [43], suggesting that BIK1 is a convergent component of both BAK1-dependent and BAK1-independent PRRtriggered signaling pathways. Whether XA21 interacts with OsRLCK185 remains to be tested. Together, these data nevertheless already indicate an overall conservation in the molecular mechanisms underlying the BIK1-PRR interactions between monocots and dicots. EFR associates dynamically with Arabidopsis orthologues of XA21interacting proteins in a ligand-dependent manner The functionality of the EFR:XA21 and XA21 proteins in Arabidopsis, and the association of EFR:XA21 with known EFR signaling components suggests a large degree of conservation between XA21 and EFR signaling in monocots and dicots, respectively. Several XA21-interacting proteins have been identified in rice [26], but interestingly there is currently no evidence that their corresponding orthologues in dicots may also be involved in PRR signaling. We directly investigated whether Arabidopsis orthologues of some of these XA21 signaling components, namely XB15 and XB24, are involved in EFR signaling. XA21 associates with XB24, which possesses ATPase activity and is a negative regulator of XA21 function [50]. A mutation in the ATPase active site of rice XB24 abolished its activity [50]. Phylogenetic analysis of XB24 revealed that orthologues are present as a single copy gene in every plant genome analyzed, including moss, monocots and dicots (Fig. 4A). Interestingly, Arabidopsis XB24 (AtXB24) has a deletion of the ATPase signature present in rice XB24 (S5 Fig.). In addition, rice XB24 appears to be the only XB24 homologue to contain this motif, suggesting that ATPase activity may not be critical for the in vivo function of XB24 in some species. The association between the previously uncharacterized Arabidopsis orthologue AtXB24 (At2g35900) and EFR was assessed. Association of HA-tagged AtXB24 with EFR-GFP was observed after transient co-expression in N. benthamiana and co-immunoprecipitation (Fig. 4B,C). Notably, a reduction of the association of AtXB24 was observed following elf18 treatment, indicating a partial dissociation from EFR. This pattern of dissociation is similar to that observed in rice with the interaction between XA21 and XB24, where interaction was only observed in uninfected tissue and XB24 dissociated within 24 hours of Xoo infection [50]. This indicates that AtXB24 and rice XB24 are dynamically related in their association kinetics with RKs. XB15 is a PP2C that dephosphorylates XA21 in vitro and negatively regulates XA21-dependent immunity to Xoo [51]. XB15 is related to the Arabidopsis POLTERGEIST (POL) family of PP2Cs [51]. Of this family, XB15 is most closely related to POLTERGEIST LIKE 4 (PLL4; AT2G28890) and 5 (PLL5; AT1G07630) (Fig. 4D), with percentage identity of 59% and 58%, respectively (S6 Fig.). PLL2 (AT5G02400) and 3 (AT3G09400) are less closely related and their expression is either not observed or restricted to roots [68]. It was shown previously that XB15, POL and POLTERGEIST LIKE 1 (PLL1) localize to the plasma membrane via myristoylation and palmitoylation [51,69]. PLL4 and PLL5 are almost identical to POL and PLL1 in the region required for membrane association. Therefore, PLL4 and PLL5 were selected as the most probable orthologues of XB15. We tested if PLL4 and PLL5 could associate with EFR by co- PLOS Pathogens | DOI:10.1371/journal.ppat.1004602 January 21, 2015 10 / 22 Conservation of Immune Signaling between Monocot and Dicot PRRs Figure 4. EFR associates dynamically with the A. thaliana orthologues of OsXB15 and OsXB24 in planta. A,D. Phylogenetic analysis of XB24 and XB15 in rice and Arabidopsis. OsXB24 is a single gene in all plant genomes analyzed. OsXB15 most closely relates to AtPLL4 and AtPLL5. Distance trees are based on protein sequences aligned with MUSCLE (produced with PHYLIP, using PROTDIST and NEIGHBOR). Accession numbers for sequences used can be found in S4 Table and alignments in S1 and S2 Figs. B,E. Co-immunoprecipitation of AtXB24 or AtPLL4 and AtPLL5 (A. thaliana orthologues of OsXB15) with EFR. EFR:GFP and HA-tagged AtXB proteins were co-expressed transiently in N. benthamiana. Leaves were treated with 100 nM elf18 (+) or mock treated with water (−) for 10 min. Immunoprecipitation (IP) was performed with GFP-Trap agarose beads. Western blots were performed with HRPconjugated anti-GFP or anti-HA. Experiments were performed at least three times with similar results. C, F. Quantitation of data presented in B and E. Values are the average ratio of AtXB24:HA, PLL4:HA or PLL5:HA to EFR:GFP ±SE (n = 3). Asterisks indicate statistical significance of mock compared to elf18 treated, p < 0.01 (Student’s t-test). nd is not determined. doi:10.1371/journal.ppat.1004602.g004 PLOS Pathogens | DOI:10.1371/journal.ppat.1004602 January 21, 2015 11 / 22 Conservation of Immune Signaling between Monocot and Dicot PRRs immunoprecipitation following transient expression in N. benthamiana. HA-tagged PLL4 and PLL5 expressed well, and both PLL proteins precipitated with EFR in the absence of elf18 treatment (Fig. 4E,F). Interestingly, we observed a reduction in the association of both PLL proteins with EFR following 10 min elf18 treatment (Fig. 4E,F). This result indicates that activation of EFR results in dissociation of PLL4 and PLL5 from the complex. Notably, the kinetics of association between XB15 to XA21 in rice seems different as the complex could only be observed 12–24 hours post-infection with Xoo [51]. However, since XA21 protein levels similarly increase during infection [51], it is difficult to determine the relative association of XB15 to XA21. Based on our results, it will be interesting upon identification of the XA21 ligand to test if XA21 activation also leads to XB15 dissociation. The Arabidopsis XB15 orthologues PLL4 and PLL5 play a role in EFRmediated immunity The dynamic association between EFR and the Arabidopsis XB15 and XB24 orthologues (namely PLL4, PLL5 and AtXB24, respectively) indicated that they play a potential role in EFR-mediated immunity. To test this hypothesis we obtained two independent T-DNA insertion lines for PLL4, PLL5 and AtXB24 each with altered transcript levels (S7 Fig.). Morphologically, Atxb24 lines were indistinguishable from wild-type, however pll5-1 exhibits darker rosette leaves that are also altered in shape and curling, as previously described [68] (S8 Fig.). The lack of morphological alterations in pll5-3 is presumably due to the residual expression of PLL5 in this line (S7 Fig.). We also generated a double pll4-1 pll5-1 mutant by crossing. The pll4-1 pll5-1 has a morphology closer to wild-type plants, suggesting some degree of antagonism between PLL4 and PLL5 during development as previously described [68]. To test the involvement of PLL4, PLL5 and AtXB24 in EFR-mediated signaling we determined the extent of the ROS burst, MAPK activation, seedling growth inhibition and induced resistance following treatment with elf18 in these mutants (Figs. 5, S9 and S10). The elf18induced ROS burst was slightly altered in Atxb24-2 (Fig. 5A). While this effect was minor and not statistically significant, it was reproducibly observed in 5 out of 6 independent experiments. Atxb24-1 were wild-type for ROS generation; this is presumably due to the presence of remaining AtXB24 transcripts in this mutant (S7A,B Fig.). Both Atxb24-1 and Atxb24-2 responded similar to wild-type Col-0 plants in the MAPK activation, induced resistance and seedling growth inhibition assays (Figs. 5 B,C, S9 and S10. In rice, reduction in XB24 transcript levels only improves resistance to Xoo in the presence of XA21 and XB24 overexpression diminishes resistance via destabilization of XA21 following activation [50]. The reduced expression in the knock-down line Atxb24-2 only slightly reduced elf18-induced ROS burst; while on the basis of XB24 function in rice one may have expected the opposite. In addition, we did not observe a significant reduction in EFR levels upon XB24 co-expression or elf18 treatment (Fig. 4B). Given the lack of ATPase motif in the Arabidopsis homologue (S5 Fig.), it may be that the mechanism of action is different and AtXB24 is acting a positive regulator (at least of the elf18-induced ROS burst) in Arabidopsis, rather than a negative regulator, as it does in rice. The single pll mutants did not show any significant alterations in elf18-induced ROS burst, MAPK activation, seedling growth inhibition or induced resistance (S9–S10 Figs.). The double mutant was also unaltered in elf18-induced MAPK activation (S10 Fig.). However, the double pll4-1 pll5-1 exhibited slight increase in the amplitude of ROS burst in response to elf18 treatment, which also peaked earlier than in wild-type leaves (Fig. 5D). This effect was reproducibly observed in 6 out of 7 independent experiments, although it is not statistically significant. Elf18-induced seedling growth inhibition was also altered in pll4-1 pll5-1 seedlings, being *10% PLOS Pathogens | DOI:10.1371/journal.ppat.1004602 January 21, 2015 12 / 22 Conservation of Immune Signaling between Monocot and Dicot PRRs Figure 5. AtXB4 and PLL4/5 play a role in EFR-mediated signaling. A,D. Elf18-induced ROS burst. Leaf discs were treated with 100nM elf18 treatment. Values are averages ±SE (n = 3, representing independent biological experiments with 12 samples in each experiment). Plant lines were not statistically different, p > 0.05 (two-way ANOVA). The reduced ROS burst in Atxb24-2 and pll4-1 pll5-1 lines was observed in 5/6 and 6/7 experiments, respectively. B, E. Seedling growth inhibition in response to increasing dose of elf18. Seedlings were grown in the presence of elf18 and seedling fresh weight recorded 10 days post-treatment. Values are averages ±SE (n = 12). Plant lines were not statistically different, p > 0.05 (two-way ANOVA). The experiment was repeated three times with similar results. C,F. Elf18-induced resistance to Pto DC3000. Plants were pre-treated with mock or 1 μM elf18 for 24 h before infection with Pto DC3000 (OD600 = 0.0002). Bacterial populations were scored at 2 dpi. Values are averages ±SE (n = 4). Letters indicate statistically significant differences from Tukey’s HSD mean separation (p < 0.05). Infections were repeated at least four times with similar results. doi:10.1371/journal.ppat.1004602.g005 more sensitive to 1 and 10 nM elf18 than Col-0 seedlings (Fig. 5E). Notably, the pll4-1 pll5-1 plants exhibited improved resistance to Pto already in the absence of elf18 treatment, restricting by *1 log bacterial growth compared to wild-type Col-0 (Fig. 5F). Elf18 treatment resulted in a further reduction in bacterial population, although this was to a lesser degree than in PLOS Pathogens | DOI:10.1371/journal.ppat.1004602 January 21, 2015 13 / 22 Conservation of Immune Signaling between Monocot and Dicot PRRs wild-type (Fig. 5F). These results revealed that PLL4 and PLL5 act as negative regulators of immunity in Arabidopsis, similarly as XB15 in rice. In rice, a transposon-insertional XB15 mutant exhibits constitutive cell death lesions, enhanced XA21-mediated resistance and induction of defense gene expression. We did not observe cell death or constitutive PR1 or MAPK activation in pll4-1 pll5-1 plants under our conditions (S10 Fig.), suggesting that there may be a greater level of redundancy in this regulation system in Arabidopsis than in rice, or that the outputs of the PRRs, which they control, do not induce cell death. PLL4 and PLL5 may negatively regulate several PRRs in addition to EFR, such that in pll4-1 pll5-1 the observed increased resistance may be a result of the cumulative hyper-activation of multiple PRRs by Pto. Such promiscuity could account for the morphological phenotypes observed in pll5-1, as PLL5 may also associate with several RKs involved in development, as previously suggested for POL and PLL1 [70]. However, these morphological alterations most likely do not account for the immune defects observed, since pll5-1 is more severely altered in development but does not display any immunity-related phenotype (S8–S10 Figs.). Conclusion The transfer of PRRs between plant species is an approach by which durable broad-spectrum disease resistance could be achieved [17,18]. Thus, there is a great interest in understanding the mechanisms of PRR-mediated resistance and how these could be applied biotechnologically. The recent successful transfer of PRRs across plant families [21,23–25,71] indicates that signaling components acting downstream of PRRs are conserved, and thus that transferred PRRs can simply ‘plug-in’ into these pathways somehow as ‘antennae’ being newly connected to a preexisting communication network. We have here used two related PRRs from the same subfamily of LRR-RKs (XII), namely Arabidopsis EFR and rice XA21, to test directly the extent of conservation in PRR-mediated signaling between two different plant clades. Although monocots and dicots diverged over 150 million years ago [72], we revealed here that the XA21 intracellular domain is functional at activating immune signaling in Arabidopsis in response to the PAMP elf18 when fused to the extracellular domain of EFR, indicating that the XA21 intracellular domain is able to interact biochemically with similar components as EFR in Arabidopsis. Indeed, we demonstrated that the EFR:XA21 chimera associates with the co-receptor BAK1 and the cytoplasmic kinase BIK1, which are both common interactors and regulators of multiple PRRs in Arabidopsis. Notably, the association of the chimera with BAK1 could only be observed upon elf18 treatment, while BIK1 dissociated from the chimera after elf18 perception. These ligand-dependent dynamic associations clearly indicate the functional biochemical interactions between the EFR:XA21 chimera and these key Arabidopsis immune components. Motivated by the ability of the XA21 intracellular domain to channel immune signaling in Arabidopsis, we hypothesized that EFR-mediated signaling may employ signaling components that are orthologous to those used by XA21 in rice. Indeed, we showed that the putative ATPase AtXB24 and the PP2Cs PLL4 and PLL5 associate with EFR and that elf18 perception leads to the partial dissociation of these complexes. AtXB24 appeared to be a positive regulator of elf18-triggered ROS burst in Arabidopsis, but did not affect other responses triggered by elf18, such as seedling growth inhibition and induced resistance to Pto DC3000. In contrast, PLL4 and PLL5 are partially redundant in negatively regulating elf18-induced ROS burst and seedling growth inhibition, as well as immunity to Pto DC3000. It will be interesting in the future to decipher the mechanisms by which these proteins regulate EFR. Notably, in parallel to this study, we recently demonstrated that EFR is functional in rice, confers slight enhanced resistance to hypovirulent bacterial strains, and recruits the same PLOS Pathogens | DOI:10.1371/journal.ppat.1004602 January 21, 2015 14 / 22 Conservation of Immune Signaling between Monocot and Dicot PRRs signaling components as XA21 (Schwessinger et al., submitted). Together, these studies provide experimental evidence for the overall conservation of immune signaling between monocots and dicots, which paves the way for further PRR transfers between these clades. Our work also illustrates the usefulness of comparative studies between monocots and dicots, as well as between plant models and crops, to reveal novel immune components. Materials and Methods Plant material and growth conditions Arabidopsis thaliana was grown in soil under short day condition (10 h light) at 20–22°C. Nicotiana benthamiana plants were grown in soil at 22°C with a photoperiod of 16 h. T-DNA insertion lines were obtained from NASC and were genotyped using the primers described in S2 Table. Chemicals Elf18 peptide was obtained from Peptron Inc. (Korea). Vector construction Full length EFR:GFP was cloned by fusing GFP to a cDNA clone of EFR, using overlap extension PCR of amplified DNA fragments 1 and 2 (S1 Table). EFR:GFP was cloned into pENTR/ D-TOPO and transferred into pEARLEYGATE100 by Gateway cloning. XA21:GFP was cloned as a NarI/NheI PCR fragment, generated from pUBI:XA21:GFP plasmid [73], into pBIN19g which had been digested with ClaI and XbaI. Chimeric receptor constructs were produced by overlap extension PCR using respective PCR-amplified DNA fragments (S1 Table). Chimeric PCR products were cloned by Gateway cloning into pEARLEYGATE100, for EFR:XA21:GFP or pEARLEYGATE103, for XA21:EFR:GFP. Cytoplasmic deletion constructs were produced by PCR from EFR or EFR:XA21 cDNA and cloning into pENTR/D-TOPO then subcloning into pEARLEYGATE103 by Gateway cloning. 35S promoter HA-tagged PLL4, PLL5 and AtXB24 constructs were amplified by PCR from Arabidopsis leaf cDNA and cloned using Gateway cloning into pGWB14. All constructs were sequenced to ensure they were correct and no mutations had been introduced by PCR. PCR primers used for cloning and generation of chimeras can be found in S1 Table. Agrobacterium-mediated transient expression Transient expression was performed by diluting an overnight culture A. tumefaciens GV3101 containing the appropriate expression plasmid to OD600 = 0.2 in 10mM MES (pH5.6), 10mM MgCl2 and 100μM acetosyringone. Four-week-old N. benthamiana leaves were syringe infiltrated. Samples were collected for analysis two days later. ROS burst Oxidative burst measurements were performed as described previously [39]. Leaf discs were taken from 4-week-old Arabidopsis plants or N. benthamiana 2 days after transient infection with Agrobacterium. Seedling growth inhibition Growth inhibition measurements were performed as described previously [39]. PLOS Pathogens | DOI:10.1371/journal.ppat.1004602 January 21, 2015 15 / 22 Conservation of Immune Signaling between Monocot and Dicot PRRs MAPK activation Leaf discs were incubated overnight in water then treated with 100nM elf18 and samples were frozen in liquid nitrogen. Samples to test for a potential constitutive activation of MAPKs were frozen directly from the plant. Proteins were extracted by grinding samples and boiling for 5 min in Laemmli sample buffer. Protein extracts were run on 10% SDS PAGE gel and transferred onto PVDF membrane. Western blots were performed with anti-p44/42 MAPK (Cell Signaling Technology) and HRP conjugated anti-rabbit antibodies (Sigma). Quantitative RT-PCR Quantitative RT-PCR was performed as described previously [74], with the exception that adult leaves of soil-grown plants were used. Bacterial infections Induced resistance was performed by syringe infiltrating 1μM elf18, or water for mock treatments, into 4-week-old Arabidopsis leaves. The following day pretreated leaves were syringe infiltrated with Pto DC3000 suspended in 10mM MgCl2 at an OD600 of 0.0002. Samples were obtained 2 days post infection to determine bacterial population. Spray inoculations were performed by spraying Pto DC3000 COR− suspended in 10mM MgCl2 and 0.02% Silwet L-77 at an OD600 = 0.2. Plants were covered and samples were collected 3 days post infection to determine bacterial populations. Agrobacterium infections of Arabidopsis plants were performed as described previously [13]. Samples for colorimetric GUS staining and quantitative MUG assays were taken 3 days post infection. Immunoprecipitation Leaf tissue was treated with 100nM elf18 by vacuum infiltration for 10min, then frozen in liquid nitrogen. Protein was extracted from N. benthamiana or Arabidopsis in 5 ml per g of tissue of extraction buffer (150 mM Tris-HCl pH 7.5; 150 mM NaCl; 10% glycerol; 1% IGEPAL CA630; 10mM DTT; protease inhibitor cocktail (Sigma)). For N. benthamiana 1% PVPP was added. Extracts were centrifuged at 10000g for 15 min and filtered through Miracloth. GFPTrap agarose beads (manufacturer) were added and extracts were incubated for 3 hr at 4°C. Beads were washed with extraction buffer without DTT and containing 0.1% IGEPAL CA630. Protein was eluted with SDS loading buffer by boiling for 4 min. Western blotting was performed with HRP conjugated anti-GFP (Santa Cruz) or anti-HA (Roche) or unconjugated anti-BAK1 described by Schwessinger et al. [56]. Phylogenetic analysis Protein sequences were obtained from NCBI and aligned using MUSCLE[75]. Accession numbers of these sequences can be found in S4 Table and alignments used for phylogenetic analysis are shown in S1 and S2 Figs. Phylogenetic analysis was performed using PHYLIP [76]. Trees were constructed using PRODIST and NEIGHBOR. Statistical analysis Graphpad prism was used to conduct statistical analysis. One-way analysis of variance was performed with post-hoc Turkey’s HSD comparison. Alternatively Student’s t-tests were performed for paired comparisons. PLOS Pathogens | DOI:10.1371/journal.ppat.1004602 January 21, 2015 16 / 22 Conservation of Immune Signaling between Monocot and Dicot PRRs Supporting Information S1 Fig. Localization of EFR, XA21 and chimeric receptors. Confocal microscopy of GFP tagged EFR (A), XA21 (B), EFR:XA21 (C) and XA21:EFR (D) in N. benthamiana. Arrows indicate the perinuclear endoplasmic reticulum. All images are taken at the same scale. (TIF) S2 Fig. Marker gene expression in EFR and EFR:XA21 transgenic Arabidopsis. Quantitative RT-PCR was used to determine PR1 (A), FRK1 (B), PHI1 (C) and NHL10 (D) in response to elf18 treatment. Treatments were performed with 100 nM elf18, except for PR1 expression where 1 μM was used. Expression is relative to U-box expression and was calculated by the comparative CT method. Values are averages ±SE (n = 6). Asterisks indicate significantly different values as compared to efr-1 at p <0.05 (one-way ANOVA). (TIF) S3 Fig. Expression of EFR, XA21, EFR:XA21 or XA21:EFR does not lead to constitutive MAPK activation or PR1 expression. A. MAPK activity of untreated transgenic lines. MAPK activation in Col-0 by 15 min flg22 treatment (flg22) is shown as a positive control. Western blots were performed with anti-p44/42 MAPK antibodies. Experiments were performed three times with similar results. B. PR1 expression in untreated transgenic lines. Expression is relative to U-box expression and was calculated by the comparative CT method. Values are averages ±SE (n = 6). No significant difference was seen between lines, p < 0.05, as determined by one-way ANOVA. (TIF) S4 Fig. BAK1 interaction is dependent on the extracellular domain. A. Co-immunoprecipitation of transiently-expressed EFRΔcyt:GFP and EFR:XA21Δcyt::GFP with BAK1:HA in N. benthamiana. Leaves were treated with 100 nM elf18 (+) or mock treated with water (−) for 10 min. Immunoprecipitation (IP) was performed with GFP-Trap agarose beads. Western blots were performed with HRP-conjugated anti-GFP and anti-HA antibodies. B. Elf18-induced ROS production in N. benthamiana expressing EFR:GFP, EFR:XA21:GFP, EFRΔcyt:GFP and EFR:XA21Δcyt:GFP. ROS production is represented as the total of photons emitted during 40 min after treatment with 100 nM elf18. Values are averages ±SE (n = 12). Asterisks indicate statistical difference from GUS transformed leaves, p < 0.0001 (Student’s t-test). Experiments were performed at least twice with similar results. (TIF) S5 Fig. Multiple alignment of XB24 orthologues. Alignment of XB24 homologues used to create the phylogenetic tree in Fig. 4A. The ATPase motif of XB24 is highlighted in red. Accession numbers are listed in S4 Table. (DOCX) S6 Fig. Multiple alignment of XB15 orthologues. Alignment of the XB15/POL homologues used to create the phylogenetic tree in Fig. 4D. Accession numbers are listed in S4 Table. (DOCX) S7 Fig. Description of Atxb24, pll4 and pll5 T-DNA mutants. A,C,E. Schematic representation of the T-DNA insertion sites in AtXB24, PLL4 and PLL5. Arrows above the genes indicate the positions of primers used for RT-PCR. B,D,F. Semi-quantitative RT-PCR of Atxb24, pll4 and pll5 T-DNA insertion lines. Primers are listed in S3 Table. (TIF) PLOS Pathogens | DOI:10.1371/journal.ppat.1004602 January 21, 2015 17 / 22 Conservation of Immune Signaling between Monocot and Dicot PRRs S8 Fig. Phenotype of Col-0, pll4, pll5, pll4 pll5, and Atxb24 adult plants. Plants were photographed 6 weeks after sowing. (TIF) S9 Fig. pll4 and pll5 single mutants phenotypes. A. Elf18-induced ROS burst. Leaf discs were treated with 100nM elf18 treatment. Values are averages ±SE (n = 3, representing independent biological experiments with 12 samples in each experiment). Plant lines were not statistically different, p > 0.05 (two-way ANOVA). B. Seedling growth inhibition in response to increasing dose of elf18. Seedlings were grown in the presence of elf18 and seedling fresh weight recorded 10 days post-treatment. Values are averages ±SE (n = 12). Plant lines were not statistically different, p > 0.05 (two-way ANOVA). The experiment was repeated three times with similar results. C. Elf18-induced resistance to Pto DC3000. Plants were pre-treated with mock or 1 μM elf18 for 24 h before infection with Pto DC3000 (OD600 = 0.0002). Bacterial populations were scored at 2 dpi. Values are averages ±SE (n = 4). Letters indicate statistically significant differences from Tukey’s HSD mean separation (p < 0.05). Infections were repeated at least four times with similar results. (TIF) S10 Fig. MAPK activation and basal PR1 expression is not altered in pll4, pll5 or Atxb24 mutants. A. MAPK activation in pll4, pll5, pll5-1 pll4-1 and Atxb24 insertion lines after elf18 treatment. Western blots were performed simultaneously and exposed for the same time. Western blots were performed with anti-p44/42 MAPK antibodies. Even loading is demonstrated by Coomassie brilliant blue (CBB) staining. The experiment was repeated twice with similar results. B. Basal PR1 expression in pll4, pll5 and Atxb24 lines. Expression is relative to U-box expression and was calculated by the comparative CT method. Values are averages ±SE (n = 6). No significant difference was seen between lines, p < 0.05, as determined by one-way ANOVA. (TIF) S1 Table. Primers used for cloning. (XLSX) S2 Table. Primers used for genotyping. (XLSX) S3 Table. Primers used for RT-PCR. (XLSX) S4 Table. Sequences used for phylogenetic analyses. (XLSX) Acknowledgments We thank past and present members of the Zipfel laboratory for useful discussion, The Sainsbury Laboratory transformation service and the John Innes Centre horticultural service for their excellent work. We thank Benjamin Schwessinger for critical reading and helpful discussion prior to submission. Author Contributions Conceived and designed the experiments: NH VN CZ. Performed the experiments: NH VN. Analyzed the data: NH VN CZ. Contributed reagents/materials/analysis tools: PCR. Wrote the paper: NH CZ. PLOS Pathogens | DOI:10.1371/journal.ppat.1004602 January 21, 2015 18 / 22 Conservation of Immune Signaling between Monocot and Dicot PRRs References 1. Lehti-Shiu MD, Shiu SH (2012) Diversity, classification and function of the plant protein kinase superfamily. Philos Trans R Soc Lond B Biol Sci 367: 2619–2639. PMID: 22889912 2. Gao LL, Xue HW (2012) Global Analysis of Expression Profiles of Rice Receptor-Like Kinase Genes. Molecular Plant 5: 143–153. doi: 10.1093/mp/ssr062 PMID: 21765177 3. Hwang SG, Kim DS, Jang CS (2011) Comparative analysis of evolutionary dynamics of genes encoding leucine-rich repeat receptor-like kinase between rice and Arabidopsis. Genetica 139: 1023–1032. doi: 10.1007/s10709-011-9604-y PMID: 21879323 4. 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Barr et al. BMC Public Health (2020) 20:927 https://doi.org/10.1186/s12889-020-08987-w RESEARCH ARTICLE Open Access Sociodemographic inequities associated with participation in leisure-time physical activity in sub-Saharan Africa: an individual participant data meta-analysis Anna Louise Barr1, Uttara Partap1,2, Elizabeth H. Young1,2, Kokou Agoudavi3, Naby Balde4, Gibson B. Kagaruki5, Mary T. Mayige6, Benjamin Longo-Mbenza7,8, Gerald Mutungi9, Omar Mwalim10, Chea S. Wesseh11, Silver K. Bahendeka12,13, David Guwatudde14, Jutta M. Adelin Jørgensen15, Pascal Bovet16,17, Ayesha A. Motala18 and Manjinder S. Sandhu1* Abstract Background: Leisure-time physical activity (LTPA) is an important contributor to total physical activity and the focus of many interventions promoting activity in high-income populations. Little is known about LTPA in subSaharan Africa (SSA), and with expected declines in physical activity due to rapid urbanisation and lifestyle changes we aimed to assess the sociodemographic differences in the prevalence of LTPA in the adult populations of this region to identify potential barriers for equitable participation. Methods: A two-step individual participant data meta-analysis was conducted using data collected in SSA through 10 population health surveys that included the Global Physical Activity Questionnaire. For each sociodemographic characteristic, the pooled adjusted prevalence and risk ratios (RRs) for participation in LTPA were calculated using the random effects method. Between-study heterogeneity was explored through meta-regression analyses and tests for interaction. Results: Across the 10 populations (N = 26,022), 18.9% (95%CI: 14.3, 24.1; I2 = 99.0%) of adults (≥ 18 years) participated in LTPA. Men were more likely to participate in LTPA compared with women (RR for women: 0.43; 95%CI: 0.32, 0.60; P < 0.001; I2 = 97.5%), while age was inversely associated with participation. Higher levels of education were associated with increased LTPA participation (RR: 1.30; 95%CI: 1.09, 1.55; P = 0.004; I2 = 98.1%), with those living in rural areas or self-employed less likely to participate in LTPA. These associations remained after adjusting for time spent physically active at work or through active travel. Conclusions: In these populations, participation in LTPA was low, and strongly associated with sex, age, education, self-employment and urban residence. Identifying the potential barriers that reduce participation in these groups is necessary to enable equitable access to the health and social benefits associated with LTPA. Keywords: Leisure-time physical activity, Physical activity, Sub-Saharan Africa, Occupational physical activity, Active travel, Global physical activity questionnaire, Recreation, Equity, Urbanisation, Mechanisation * Correspondence: mss31@cam.ac.uk 1 Department of Medicine, University of Cambridge, Cambridge, UK Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Barr et al. BMC Public Health (2020) 20:927 Background Physical activity can be undertaken within several domains of an individual’s life, with work, travel and leisure-time the most commonly defined. In many highand middle-income countries, increasing mechanisation and the rise in sedentary employment have led to notable declines in the time spent in occupational physical activity [1, 2]. Likewise, urbanisation and increasing dependence on motor vehicles has led to a reduction in the amount of physical activity accumulated through active travel [3]. Conversely, many of these same countries have observed an increase in the contribution of leisure-time physical activity (LTPA) to total physical activity [1]. Globally, LTPA and its associated market is worth billions, [4] and is an important target for public health interventions attempting to reduce the epidemic of insufficient physical activity amongst populations [5]. Importantly, a number of prospective studies and metaanalyses have indicated that LTPA may have greater health benefits compared with other domains of physical activity [6–8]. However, not all groups are engaged in regular LTPA: women, older adults, and low-income or less educated populations are often less likely to participate [9, 10]. Many high-income countries (HICs) are working hard to identify and tackle the social, economic and environmental barriers which prevent these groups from participating in regular LTPA, and to ensure equitable access to the associated health and social benefits [11–13]. By contrast, knowledge of the physical activity patterns of populations in sub-Saharan Africa (SSA) is limited. Africa is the fastest urbanising continent, [14] and in recent decades has experienced rapid lifestyle changes, mirroring those observed in higher income countries. A recent review illustrated that for sub-Saharan African countries with lower levels of physical activity, the contribution of occupational physical activity to total physical activity was reduced, while the contribution of active travel increased [15]. The contribution of LTPA was low across all countries [15]. However, this review did not estimate the prevalence of participation across the different domains of physical or assess the differences in participation across population subgroups. With increasing economic development, the contribution of LTPA to total physical activity is likely to rise in sub-Saharan African populations; however, currently little is known about those participating in LTPA. Using data from 10 diverse populations across SSA, we sought to address the current gap in understanding by assessing the prevalence of participation in LTPA, and the associated sociodemographic factors, to help identify the groups at risk of exclusion and potential barriers to participation within the African context. Page 2 of 13 Methods Survey methods As part of the ongoing collaborative work of the African Partnership for Chronic Disease Research, individual participant data (IPD) from 10 large-scale adult population surveys were collated for the purpose of these analyses. These surveys were conducted in nine countries: Democratic Republic of Congo (DRC), [16] Guinea, [17] Kenya, [18] Liberia, [19] Seychelles, [20] South Africa, [21] United Republic of Tanzania (consisting of Tanzania and Zanzibar), [22, 23] Togo, [24] and Uganda [25]. All studies utilised or were consistent with the WHO STEPwise approach to noncommunicable disease risk factor surveillance tool (STEPS), which includes the Global Physical Activity Questionnaire (GPAQ) [26]. The methods of each survey are described in greater detail elsewhere [16, 17, 19–25, 27]. Each study employed a multi-level sampling strategy (Supplementary Table S1), and was designed to be representative of the national or subnational populations from which they were drawn. Surveys were conducted through face-to-face interviews, with all surveys apart from Liberia having responses greater than 70.0% (Supplementary Table S1). Definition of exposure and outcomes Age, in years, was recoded into six categories: 18–24, 25–34, 35–44, 45–54, 55–64, and 65 and above. Education was grouped into four categories based on the highest education level completed: primary education not completed, primary education completed, secondary education completed, and tertiary education completed (Supplementary methods). Employment status was recategorised from nine to four categories (Supplementary methods): public or private employees; self-employed; non-income workers; and no occupation. Participants’ height and weight measurements were used to calculate their body-mass index (BMI). BMI was categorised into four categories based on standard cut-offs: underweight (BMI: < 18.5 kg/m2); healthy weight (BMI: 18.5–24.9 kg/ m2); overweight (BMI: 25.0–29.9 kg/m2); and obese (BMI: ≥ 30.0 kg/m2). Six studies collected data on urban or rural residence. Physical activity variables were cleaned according to the GPAQ guidelines [28]. Insufficient physical activity was defined as undertaking less than 600 metabolic equivalent (MET) minutes of physical activity per week, [29] while participation in physical activity was defined as undertaking > 0 MET-minutes of physical activity per week. Participation in LTPA, occupational physical activity or active travel was defined as undertaking > 0 METminutes of physical activity within these domains. Statistical analyses We conducted a two-step IPD meta-analysis, analysing each dataset separately to obtain study-level estimates, Barr et al. BMC Public Health (2020) 20:927 before combining them using random effects models of meta-analysis. Participants were included if individuals were aged 18 years and above and had complete information on sex, age, education, employment, BMI, physical activity and sampling units. All analyses were conducted using Stata/SE 14.2 (StataCorp, Texas). Adjusted prevalence estimates of the sociodemographic characteristics investigated were calculated for each individual study population using mixed effects Poisson regression models with robust standard errors, adjusted for sex, age and clustering at each level of sampling (Supplementary Table S2). Adjusted prevalence estimates for participation in physical activity within each domain overall, and for each sociodemographic group, were then calculated for each individual study using mixed effects Poisson regression models with robust standard errors, adjusted for sex, age and clustering at each level of sampling. Using the metaprop Stata command, which is specifically designed to pool proportions, pooled adjusted prevalence estimates for sociodemographic characteristics and domain participation were calculated using a random effects model [30]. The metaprop command requires a total for each sub-group (N) and a number of the participants within that sub-group with the condition (n). The predicted n was calculated by multiplying the individual country adjusted prevalence estimates against the N for that study–the total number of participants in each study with complete data. The study-specific confidence intervals were calculated using the exact method [30]. The outcomes were normalised using the Freeman-Tukey double arcsine transformation and the pooled adjusted prevalence estimates were computed using the DerSimonian and Laird method [31]. The confidence intervals of the pooled adjusted prevalence estimates were calculated using the Wald method [30]. Poisson regression models, with robust standard errors, were fitted to obtain study-specific risk ratios (RRs) for participation in each domain of physical activity. Mixed effects accounted for the clustered nature of the data. Fullyadjusted models were adjusted for sex, age, education, employment and BMI. The pooled RRs were calculated for each sociodemographic characteristic using a weighted average of the log-adjusted RR, allowing for random effects using the DerSimonian and Laird method [31]. Sensitivity analyses were conducted to check whether a single study substantially influenced the pooled RRs, by excluding each study from the pooled analyses in turn and comparing results with and without the study in question. In supplementary analyses, we investigated whether adjustment for participation in physical activity at work or through travel, or the volume of physical activity accumulated in each domain, affected observed associations with participation in LTPA. Page 3 of 13 The I2 statistic was used to assess the heterogeneity between study-specific estimates e.g. pooled adjusted prevalence estimates and RRs [32]. We considered values of ≥75% to indicate high heterogeneity, values < 75 and > 25% to indicate moderate heterogeneity and values of ≤25% to indicate low heterogeneity [33]. Effect modification was assessed by stratifying the fully-adjusted model by sex, education and residence location (urban/rural). We also assessed whether there was statistical evidence of interaction between sex and all other covariates, education and all other covariates, and residence location and all other covariates, regarding their association with participation in LTPA. This was done by including an interaction term within the fully-adjusted model and pooling the log RR for the interaction term using the method of DerSimonian and Laird described above. Finally, random effects meta-regression was used to explore the influence of study design and population characteristics, listed in Table 1, on the between-study heterogeneity for the fully-adjusted models. Additionally, as the calculated P-values using this method tend to be conservative, we conducted permutation tests based on Monte Carlo simulation, adjusting the P-values for multiple-testing [34]. Ethics Each primary study obtained ethical approvals in its respective country along with informed consent from participants. This study received ethical approval from the Human Biology Research Ethics Committee at the University of Cambridge, UK (Application No: HBREC.2015.05). Results The studies included in this IPD meta-analysis were undertaken over a period of 10 years and were a mixture of representative surveys from national and subnational populations (N = 26,022) (Table 1). The studies were conducted in countries at various stages of economic and urban development [35–38]. Women made up 59.5% (95% confidence interval (CI): 56.7, 62.3; I2 = 95.3%) of the population (Table 2). The majority of individuals were aged between 25 and 44 years. A small proportion of the population was educated to tertiary level and 38.5% (95%CI: 27.5, 50.0; I2 = 99.7%) were self-employed. Over one third of the population were classified as overweight or obese. For those studies which reported residence location, the majority of individuals lived in rural areas (60.8%; 95%CI: 48.3, 72.6; I2 = 99.7%). The pooled prevalence of insufficient physical activity was 9.5% (95%CI: 6.3, 13.4; I2 = 99.0%); however at least 94.1% (95%CI: 91.1, 96.5; I2 = 98.8%) of the population undertook some form of physical activity per week. Overall, the prevalence of participation in LTPA was 18.9% (95%CI: 14.3, 24.1; I2 = 99.0%) (Table 2). LTPA Barr et al. BMC Public Health (2020) 20:927 Page 4 of 13 Table 1 Study design and population characteristics of included population health surveys Study (reference) Study period Geographical scope Sample sizea Income group HDI (N) GDP per capita (US$) Urban population (%) DRC [13] 2005 Subnational 1502 Low 0.364 218.5 37.5 Guinea [14] 2009 Subnational 2125 Low 0.380 615.1 34.4 Togo [21] 2010 National 2051 Low 0.457 487.9 37.5 Liberia [16] 2011 National 2206 Low 0.416 379.7 48.2 Zanzibar [20] 2011 Subnational 2640 Low 0.504 733.4 28.8 Tanzania [19] 2012 National 5525 Low 0.513 820.2 29.5 Seychelles [17] 2013 National 1232 Upper-middle 0.766 14,764.9 53.2 South Africa [18] 2013 Subnational 1014 Upper-middle 0.660 6822.5 63.8 Uganda [22] 2014 National 3543 Low 0.488 702.8 15.8 Kenya [15] 2015 National 4184 Low-middle 0.555 1355.0 25.6 DRC Democratic Republic of Congo, Income group World Bank income group, HDI Human Development Index, GDP per capita Global domestic product per capita, Urban population: Percentage of the total population living in urban areas. World Bank income group, HDI, GDP per capita and urban population data relates to year of study [30–33], Zanzibar taken from data reported for Tanzania in 2011 as no regional specific data was available. a Sample size is the sample size after data cleaning was completed and those with no or invalid information on variables of interest removed participation ranged from 1.6% (95%CI: 0.7, 2.4) in DRC to 27.1% (95%CI: 24.7, 29.4) in Tanzania (Supplementary Table S3). By contrast, the pooled adjusted prevalence of participation in physical activity within the domains of work and travel were 70.1% (95%CI: 53.5, 84.4; I2 = 99.9%) and 86.2% (95%CI: 82.5, 89.5; I2 = 98.5%), respectively (Table 2 and Supplementary Figures S1-S2). A notably higher proportion of men participated in LTPA compared with women, with participation in LTPA decreasing substantially with age (Fig. 1). We also observed that the proportion of the population participating in LTPA increased with education level. A greater proportion of urban residents reported participating in LTPA compared with rural residents. By contrast, prevalence estimates were generally similar across sociodemographic groups for participation in occupational physical activity or active travel (Supplementary Figures S1-S2 and Supplementary Table S4-S5). In fully-adjusted Poisson regression analyses, men were more likely to participate in LTPA compared with women (RR for women: 0.43, 95%CI: 0.32, 0.60; P < 0.001; I2 = 97.5%; P for heterogeneity < 0.001) (Fig. 2). Urban residents were more likely to participate in LTPA compared with rural residents (RR for rural: 0.88; 95%CI: 0.80, 0.95; P = 0.005; I2 = 62.1%; P for heterogeneity = 0.022). A strong positive relationship between education and participation in LTPA was observed, while a strong inverse relationship between age and participation in LTPA was also detected. Being self-employed or having no occupation was associated with a notably reduced likelihood of participating in LTPA when compared with those in public or private employment. By contrast, few sociodemographic characteristics were strongly associated with participation in physical activity at work and through active travel in these populations (Figs. 3 and 4). Education was inversely associated with participation in occupational physical activity. Individuals who were self-employed had a slightly increased likelihood of participating in occupational physical activity when compared with public and private employees, while those with no occupation had a slightly reduced likelihood in participating in physical activity at work or through travel. Unlike LTPA, BMI was inversely associated with reduced participation in physical activity at work and through active travel. Notably, rural residents were more likely to participate in work and travel physical activity compared with urban residents. Sensitivity analyses revealed no evidence of outlier studies and the interpretation of the RRs for participation in work, travel and leisure-time physical activity remained unchanged regardless of which study was excluded from the pooled analyses (Supplementary Tables S6, S7, S8). Adjusting for participation in occupational physical activity or active travel, or both, did not notably change the RRs for participation in LTPA (Supplementary Figures S3, S4, S5, S6, S7, S8). Likewise, the RRs remained unchanged when total MET-minutes spent in occupational physical activity or active travel, or both, were included in the model (Supplementary Figures S3, S4, S5, S6, S7, S8). Due to low statistical power (n = 10), we were unable to detect substantive evidence that any of the study level or population characteristics listed in Table 1 explained the high levels of between-study heterogeneity observed in these analyses (Supplementary Tables S10, S11, S12). To further explore the association between sociodemographic characteristics and participation in LTPA, we stratified our models by sex, education and residence location. We observed evidence of a strong interaction between age and sex in the association with LTPA Barr et al. BMC Public Health (2020) 20:927 Page 5 of 13 Table 2 Pooled adjusted prevalence of sociodemographic characteristics (N = 26,022) N % (95%CI) I2 Men 10 40.1 (37.3, 42.9) 95.2% Women 10 59.5 (56.7, 62.3) 95.3% 18–24 9 13.0 (5.3, 23.4) 99.8% 25–34 10 29.9 (27.2, 32.5) 95.4% 35–44 10 22.4 (19.6, 25.3) 96.7% 45–54 10 17.0 (14.2, 20.0) 97.5% Characteristic a Sex Age groupb (years) 55–64 10 12.1 (9.5, 15.1) 97.9% 65+ 6 2.6 (0.6, 5.9) 99.2% Primary education not completed 9 30.2 (19.2, 42.4) 99.8% Primary education completed 10 25.7 (15.3, 37.9) 99.8% Secondary education completed 10 30.4 (17.8, 44.6) 99.8% Tertiary education completed 10 4.0 (2.4, 5.9) 98.2% Private or public employee 10 16.0 (9.4, 24.0) 99.6% Self-employed 10 38.5 (27.5, 50.0) 99.7% Education Employment Non-income work 10 23.6 (16.9, 31.0) 99.5% No occupation 10 9.4 (4.6, 15.7) 99.6% 10 7.8 (5.6, 10.3) 97.9% BMI (kg/m2) Underweight Healthy weight 10 53.7 (47.2, 60.2) 99.1% Overweight 10 21.0 (17.7, 24.4) 97.7% Obese 10 12.5 (8.0, 17.8) 99.3% Urban 6 39.2 (27.4, 51.7) 99.7% Rural 6 60.8 (48.3, 72.6) 99.7% 10 9.5 (6.3, 13.4) 99.0% 10 94.1 (91.1, 96.5) 98.8% Residence location†c Physical activity (MET-mins/wk) Insufficient Participation in physical activity (MET-mins/wk) Participation in physical activity (all domains) Leisure-time physical activity (LTPA) 10 18.9 (14.3, 24.1) 99.0% Occupational physical activity 10 70.1 (53.5, 84.4) 99.9% Active travel 10 86.2 (82.5, 89.5) 98.5% N: number of studies; %: Pooled adjusted prevalence; 95%CI: 95% confidence interval; I2: I2 statistic indicates proportion of variation due to heterogeneity; METmins/wk: Metabolic Equivalent minutes per week; Insufficient: < 600 MET-minutes per week; Participation in physical activity: > 0 MET-minutes of physical activity per week undertaken in all domains; Leisure-time physical activity: > 0 MET-minutes spent in leisure-time physical activity per week; Occupational physical activity: > 0 MET-minutes spent in occupational physical activity per week; Active travel: > 0 MET-minutes spent in physical activity through active travel per week; Underweight (BMI: < 18.5 kg/m2); Healthy weight (BMI: 18.5–24.9 kg/m2); Overweight (BMI: 25.0–29.9 kg/m2); Obese (BMI: ≥ 30.0 kg/m2) † Residence location: N = 20,022 Adjusted prevalence estimates are based on Poisson regression models adjusted for sex, age and clustering at each level of sampling a Adjusted prevalence estimates for sex are based on Poisson regression models adjusted for age and clustering at each level of sampling b Adjusted prevalence estimates for age group are based on Poisson regression models adjusted for sex and clustering at each level of sampling c Adjusted prevalence estimates for age group are based on Poisson regression models adjusted for sex and age Prevalence estimates may not add up to 100% due to the effects of adjustment Barr et al. BMC Public Health (2020) 20:927 Page 6 of 13 Fig. 1 Pooled adjusted prevalence of participation in leisure-time physical activity (N = 26,022). Prevalence: pooled adjusted prevalence; 95%CI: 95% confidence interval; N: number of studies; I-squared: I2 statistic indicates proportion of variation due to heterogeneity; Underweight (BMI: < 18.5 kg/m2); Healthy weight (BMI: 18.5–24.9 kg/m2); Overweight (BMI: 25.0–29.9 kg/m2); Obese (BMI: ≥ 30.0 kg/m2). Residence location: N = 20,022. Pooled adjusted prevalence estimates are based on two-step individual pooled data meta-analysis of Poisson regression models adjusted for sex, age and clustering at each level of sampling. Sex: pooled adjusted prevalence estimates for sex are based on two-step individual pooled data meta-analysis of Poisson regression models adjusted for age and clustering at each level of sampling. Age group: pooled adjusted prevalence estimates for age group are based on two-step individual pooled data meta-analysis of Poisson regression models adjusted for sex and clustering at each level of sampling. participation (P for interaction < 0.001) (Fig. 5). Additionally, rural men were less likely to participate in LTPA compared with urban men; however, no notable differences were observed between urban and rural women. Sensitivity analyses revealed that the RRs for women with no occupation in Guinea strongly influenced the pooled RR, with no notable difference between the sexes observed when Guinea was excluded from the pooled analyses. We observed no consistent evidence of an interaction between education and all other covariates, or residence location and all other covariates, after sensitivity analyses (Supplementary Figures S9, S10). Discussion Participation in LTPA amongst adults was low in these sub-Saharan African populations when compared with participation in occupational physical activity and active travel. Our findings suggest that women, older adults, lower educated individuals, those who were selfemployed, and rural residents were less likely to participate in LTPA, regardless of the physical activity undertaken in other domains. Few strong sociodemographic associations were observed for participation in occupational physical activity and active travel. With increasing urbanisation and mechanisation, concerted efforts should be made to ensure equitable access to LTPA and the associated health and social benefits. Due to the multitude of methods for measuring and analysing participation in physical activity across its various domains, direct comparisons with the literature were limited [2, 39–41]. The observed prevalence of LTPA participation was slightly lower than the participation levels reported in the few studies conducted in SSA. Although no time frame was given, 25.4% of South African adults reported participating in some form of LTPA in a nationally-representative survey [42]. In Nigeria, 26.5% of a predominantly rural population participated in vigorous LTPA, and 20.4% participated in moderate LTPA at least once a week [43]. This was considerably lower than reported participation in HICs: on average 40% of Europeans were estimated to engage in regular LTPA per week [9]. Among the few studies conducted in African populations, evidence of a difference in participation in LTPA between the sexes was mixed [39, 43–45]. However, our findings are consistent with larger, more representative studies conducted in other lowand middle-income countries and HICs which report less participation in LTPA amongst women [46–48]. Possible explanations include constraints associated with women’s traditional responsibilities of managing childcare and domestic chores, [49] a lack of interest, [42] and a shortage of safe and accessible opportunities for LTPA [50]. Barr et al. BMC Public Health (2020) 20:927 Page 7 of 13 Fig. 2 Association between sociodemographic characteristics and participation in leisure-time physical activity (N = 26,022). RR: Pooled risk ratio; 95%CI: 95% confidence interval; N: number of studies in two-step individual participant data meta-analysis; P-value: corresponds to the Z-test of significance for the pooled RR; I-squared: I2 statistic indicates proportion of variation due to between-study heterogeneity; P: corresponds to the P-value for the chi-squared test of heterogeneity. Pooled RRs calculated from two-step individual participant data meta-analysis of individual study RRs, estimated by multivariable mixed effects Poisson regression models with robust standard errors, adjusted for levels of clustering and all other covariates in the figure except residence location. Categorical variables for age, education and BMI were included in the model as continuous variables to reduce the degrees of freedom and enable model convergence. Residence location: N = 20,022. Men were the reference for women; public and private employees were the reference for employment; urban residents were the reference for rural residents The observed inverse relationship between age and participation in LTPA has previously been reported in African populations and globally [9, 39, 42, 45]. As people retire, and opportunities for physical activity at work or through travel potentially reduce, encouraging participation in LTPA through targeted and accessible programmes for older age groups may help prevent and manage non-communicable diseases (NCDs) and enable opportunities for socialising in a population which is often at risk of isolation. We observed a strong positive association between education and participation in LTPA. Elsewhere in Africa, increased education has been associated with reduced overall physical activity, [51, 52] with low educational attainment associated with less participation in LTPA in high- and middle-income populations [9, 46]. Those with higher education may be more aware of the health and social benefits of LTPA and thus motivated to spend their leisure-time physically active, particularly if their work is sedentary [53]. It is also likely that a complex inter-relationship exists between education, employment and socioeconomic status (SES) [53]. Higher levels of educational attainment are likely to increase individuals’ opportunities for higher paid employment, reducing the financial constraints which may prevent participation in LTPA. Likewise, these jobs may enable regular participation in LTPA by providing greater flexibility and independence at work [54]. A number of studies globally have observed reduced participation in LTPA amongst those with lower SES [55]. In Kenya, 4.1–6.3% of individuals living in urban slums engaged in LTPA, compared with the national average of 23.4% [27, 39, 44]. Time and financial constraints, as well as inaptitude, have been reported as perceived barriers to participation for those with low SES in Botswana, South Africa and other HICs [42, 53, 56]. In these analyses, being self-employed was associated with lower participation in LTPA and increased likelihood of participating in occupational physical activity compared with those in private or public employment. In this context, self-employment is often characterised Barr et al. BMC Public Health (2020) 20:927 Page 8 of 13 Fig. 3 Association between sociodemographic characteristics and participation in occupational physical activity (N = 26,022). RR: Pooled risk ratio; 95%CI: 95% confidence interval; N: number of studies in two-step individual participant data meta-analysis; P-value: corresponds to the Z-test of significance for the pooled RR; I-squared: I2 statistic indicates proportion of variation due to between-study heterogeneity; P: corresponds to the P-value for the chi-squared test of heterogeneity. Pooled RRs calculated from two-step individual participant data meta-analysis of individual study RRs, estimated by multivariable mixed effects Poisson regression models with robust standard errors, adjusted for levels of clustering and all other covariates in the figure except residence location. Categorical variables for age, education and BMI were included in the model as continuous variables to reduce the degrees of freedom and enable model convergence. Residence location: N = 20,022. Men were the reference for women; public and private employees were the reference for employment; urban residents were the reference for rural residents by low earnings and informal wages, with a high proportion involved in agricultural work [57]. This is consistent with findings from previous studies which observed that those in higher status occupations had higher levels of LTPA and lower levels of occupational physical activity compared with those in lower status occupations [54]. Rural participation in LTPA was notably lower compared with urban populations, while work and travel physical activity were notably higher, reflecting potential time and energy constraints on individuals. Rural populations are more likely to be involved in time-consuming agricultural work, which often requires high levels of energy expenditure, [52] thus these groups may not have the time or desire to participate in further physical activity during their limited leisure-time [42]. However, in these analyses RRs for participation in LTPA were not notably different after adjustment for participation and time spent physically active during work and travel. Thus, other factors may be associated with lower LTPA participation in rural populations, including fewer opportunities, especially in sparsely populated areas [58]. With increasing mechanisation, particularly in the agricultural sector, these individuals may be at risk of reduced occupational physical activity. Opportunities for LTPA should be available in order to maintain or increase physical activity levels in these populations. BMI was inversely associated with participation in physical activity at work and through travel but no association was observed with LTPA in these populations. In many HICs, reductions in occupational physical activity have been associated with decreases in energy expenditure [2]. There is also evidence that although LTPA has increased, it has not been to sufficient levels to compensate for the reduction in occupational physical activity and consequently total physical activity has decreased markedly [59]. This is likely to have had an impact on weight maintenance and the risk of NCDs in these populations [60–62]. While there are likely to be other factors involved, relating to economic development and associated lifestyle changes including diet, [60, 62] creating opportunities to access the additional health and social benefits of LTPA and to compensate for reductions Barr et al. BMC Public Health (2020) 20:927 Page 9 of 13 Fig. 4 Association between sociodemographic characteristics and participation in active travel (N = 26,022). RR: Pooled risk ratio; 95%CI: 95% confidence interval; N: number of studies in two-step individual participant data meta-analysis; P-value: corresponds to the Z-test of significance for the pooled RR; I-squared: I2 statistic indicates proportion of variation due to between-study heterogeneity; P: corresponds to the P-value for the chi-squared test of heterogeneity. Pooled RRs calculated from two-step individual participant data meta-analysis of individual study RRs, estimated by multivariable mixed effects Poisson regression models with robust standard errors, adjusted for levels of clustering and all other covariates in the figure except residence location. Categorical variables for age, education and BMI were included in the model as continuous variables to reduce the degrees of freedom and enable model convergence. Residence location: N = 20,022. Men were the reference for women; public and private employees were the reference for employment; urban residents were the reference for rural residents in occupational physical activity is undoubtedly an important consideration for public health officials in SSA, where the prevalence of overweight and obesity is already high [6–8, 63, 64]. To our knowledge this is the first meta-analysis using IPD from SSA to investigate participation within the three main domains of physical activity. Each study employed sampling strategies which aimed to recruit a representative sample of the national or subnational population from which they were drawn. The overall participant response was relatively high across studies. The studies included in these analyses all utilised or were consistent with the WHO STEPwise approach to non-communicable disease risk factor surveillance tool, a well-established survey tool for population surveillance which has been used in over 122 countries, [65] enabling the standardised collection of key variables. Data were collected by trained interviewers, with BMI objectively measured. Furthermore, identical methods were used to clean and analyse these data, enabling comparability of country- specific estimates and the calculation of more robust pooled estimates. Self-report questionnaires are currently the main way to measure the domains of physical activity. While the reliability of the GPAQ is high, the validity tends to be low to moderate [66, 67]. However, the outcome of interest for validity studies tends to be volume of physical activity rather than domain, thus it is likely the validity of the GPAQ to measure domain participation is higher as participants are more likely to remember participating in physical activity within a certain domain than the exact time and intensity [66, 67]. While the studies included in these analyses aimed to be representative of their national or subnational populations, these findings may not be generalisable to other African populations or reflect the current situation for the populations of older studies. The small number of studies included in these analyses also limited the power to detect sources of heterogeneity at the study level. Due to the cross-sectional nature of these data the temporality of associations and seasonal variations of participation could Barr et al. BMC Public Health (2020) 20:927 Page 10 of 13 Fig. 5 Association between sociodemographic characteristics and participation in leisure-time physical activity by sex. Men: N = 10,878; Women: N = 15,144. RR: Pooled risk ratio; 95%CI: 95% confidence interval; N: number of studies in two-step individual participant data meta-analysis; Pvalue: corresponds to the Z-test of significance for the pooled RR; I-squared: I2 statistic indicates proportion of variation due to between-study heterogeneity; P: corresponds to the P value for the chi-squared test of heterogeneity; P for interaction: corresponds to the Z-test of significance for the pooled interaction term. Pooled RRs calculated from two-step individual participant data meta-analysis of individual study RRs, estimated by multivariable mixed effects Poisson regression models with robust standard errors, adjusted for levels of clustering and all other covariates in the figure except residence location. Categorical variables for age, education and BMI were included in the model as continuous variables to reduce the degrees of freedom and enable model convergence. Residence location: N = 20,022. Public and private employees were the reference for employment; urban residents were the reference for rural residents not be assessed. Finally, there is the potential for residual confounding due to unaccounted for confounders. In 2017, 60% of African countries had an operational policy or strategy which addressed physical activity, with limited evidence of their effectiveness [68]. To maintain or increase population physical activity levels it will be important for governments and health officials to identify the barriers and motivations associated with participation in LTPA, particularly amongst those groups where participation is notably lower. Providing free recreational facilities will not automatically increase participation, as observed in Kenya [44]: instead a whole system approach is required [5]. Mass media campaigns, such as ‘This Girl Can’ in the UK and ‘Kau Mai Tonga’ in Tonga, have shown success in empowering women to engage in physical activity [11, 69, 70]. Such campaigns are a recommended NCD ‘Best Buy’ and can help shift social norms and perceived barriers to participation if designed appropriately for the African context [71]. Likewise, improving access to opportunities for physical activity throughout life will be critical to ensuring that participation in LTPA does not decline with age. Exposure to physical activity in school was an important motivator for adult women who participated in LTPA in South Africa [42]. Furthermore, positive experiences of physical activity at school and in other settings is important for building individual’s confidence to participate [11, 53]. Along with the health benefits, the opportunity to socialise through LTPA was a key motivator for older people in South Africa [42]. Tailoring physical activity programmes to meet the diverse needs and motivations of groups who are currently at risk of lower participation in LTPA will be important for encouraging and maintaining participation [5]. Likewise, investing in accessible and safe environments for physical activity will be critical for achieving equitable participation in LTPA [50, 72–74]. Furthermore, concerted efforts are needed to strengthen the surveillance infrastructure within SSA to collect longitudinal data on the physical activity patterns Barr et al. BMC Public Health (2020) 20:927 of these populations, ideally using objective measurement tools supplemented with diaries, to capture the domain in which physical activity is undertaken [75, 76]. This is important as recent studies suggest differential health benefits across physical activity domains [6–8]. Additionally, high levels of occupational physical activity have been associated with increased risk of all-cause mortality and cardiovascular events, particularly in men, [7, 77] with one study reporting that LTPA may modify these associations [78]. However, further studies are needed to understand the specific mechanisms by which these associations may be explained and to better account for residual confounding [75, 79]. Considering the high levels of occupational physical activity, the low participation in LTPA, and the increasing burden of NCDs among populations in SSA, these associations should be of particular interest to governments; however, without prospective research, associations between domains of physical activity and health outcomes cannot be explored. Finally, such surveillance platforms would enable the monitoring of trends and the evaluation of interventions to more clearly establish their effectiveness and inform scale-up strategies [79]. Conclusions This study adds to the limited evidence on LTPA participation amongst adults in SSA. We show that participation in LTPA is low in these populations, with women, older adults, less educated, self-employed and rural populations less likely to participate in LTPA. Further research is necessary to understand the personal, social and environmental barriers and motivators for participation in LTPA in these groups. Likewise, investment in surveillance infrastructure will allow us to track temporal trends in domain participation and improve the evidence base for policy development. Finally, as increasing urbanisation and mechanisation leads to declines in population physical activity levels, political and financial commitment from governments is necessary to ensure that the health and social benefits of LTPA are accessible to all [80]. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12889-020-08987-w. Additional file 1. Abbreviations BMI: Body-mass index; DRC: Democratic Republic of Congo; WHO: World Health Organization; STEPS: WHO STEPwise approach to non-communicable disease risk factor surveillance tool; GPAQ: Global Physical Activity Questionnaire; HICs: High-income countries; IPD: Individual participant data; LTPA: Leisure-time physical activity; SSA: Sub-Saharan Africa; RR: Risk ratio; MET: Metabolic equivalent; NCD: Non-communicable disease; SES: Socioeconomic status Page 11 of 13 Acknowledgments We would like to thank the surveys team who contributed to data collection in the primary studies. We are also grateful to all participants for their involvement in the original surveys. Authors’ contributions ALB and MSS conceived the idea for the manuscript. KA, NB, GBK, MTM, BLM, GM, OM, CSW, SKB, DG, JJ, PB, AAM, EHY and MSS provided individual participant data for analyses. ALB cleaned and analysed the data and was the major contributor in writing the manuscript. All authors were involved in the interpretation of the analyses, and read and approved the final manuscript. Funding This work was supported by the African Partnership for Chronic Disease Research strategic award from United Kingdom Medical Research Council (grant number MR/K013491/1); the Wellcome Trust (WT206194); and the National Institute for Health Research Cambridge Biomedical Research Centre (UK). The funders had no role in the study design, data collection, analysis, decision to publish, or preparation of this manuscript. Availability of data and materials The data that support the findings of this study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate Each primary study obtained ethical approvals in its respective country along with informed consent from participants. Following the WHO STEPwise approach to surveillance instrument, a consent form was read to each respondent and verbal consent was provided prior to the survey interview and collection of physical measurements for the Liberia STEPS survey, this was approved by University of Liberia’s Research Ethics Committee (University of Liberia Pacific Institute for Research and Evaluation). Written consent was provided by participants for all other studies included. This study received ethical approval from the Human Biology Research Ethics Committee at the University of Cambridge, UK (Application No: HBREC.2015.05). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details Department of Medicine, University of Cambridge, Cambridge, UK. 2 Wellcome Sanger Institute, Genome Campus, Hinxton, UK. 3Togo Ministry of Health, Lome, Togo. 4Department of Endocrinology and Diabetes, Donka University Hospital, Conakry, Guinea. 5National Institute for Medical Research, Tukuyu Research Centre, Tukuyu, Tanzania. 6National Institute for Medical Research, Headquarter Research Centre, Dar es Salaam, Tanzania. 7Faculty of Health Sciences, Walter Sisulu University, Mthatha, Eastern Cape, South Africa. 8 LOMO University of Research, Kinshasa, Democratic Republic of Congo. 9 Control of Non-Communicable Diseases Desk, Ministry of Health, Kampala, Uganda. 10Zanzibar Ministry of Health, Mnazi Mmoja, Tanzania. 11Ministry of Health, Monrovia, Liberia. 12Mother Kevin Postgraduate Medical School (MKPGMS), Uganda Martyrs University, Kampala, Uganda. 13St. Francis Hospital, Nsambya, Kampala, Uganda. 14Department of Epidemiology and Biostatistics, School of Public Health, Makerere University, Kampala, Uganda. 15 Department of Public Health, University of Copenhagen, Copenhagen, Denmark. 16University Center for Primary Care and Public Health (Unisanté), Lausanne, Switzerland. 17Ministry of Health, Victoria, Republic of Seychelles. 18 Department of Diabetes and Endocrinology, Nelson R. 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Organismal Biology A Journal of the Society for Integrative and Comparative Biology academic.oup.com/icb Downloaded from https://academic.oup.com/iob/article/1/1/obz029/5628227 by guest on 18 May 2024 Integrative Integrative Organismal Biology Integrative Organismal Biology, pp. 1–8 doi:10.1093/iob/obz029 A Journal of the Society for Integrative and Comparative Biology RESEARCH ARTICLE Functional Diversity of Evolutionary Novelties: Insights from Waterfall-Climbing Kinematics and Performance of Juvenile Gobiid Fishes
,1, R. Lagarde ,†, K.M. Diamond ¶ ,k and H.L. Schoenfuss
, R.M. Keeffe ,§ R.S. Bertram,¶
Department of Biological Sciences, Clemson University, Clemson, SC 29634, USA; †Hydr^ o Reunion, Z.I. Les Sables, 97427 Etang Sale, La Reunion, France; Universite de Perpignan Via Domitia - CNRS, Centre de Formation et de Recherche sur les Environnements Mediterraneens, UMR 5110, F 66860 Perpignan, France; §Department of Biology, University of Florida, Gainesville, FL 32611, USA; ¶Aquatic Toxicology Laboratory, St. Cloud State University, St. Cloud, MN 29634, USA; kENTROPIE, IRD, Universite de La Reunion, CNRS, Laboratoire d’Excellence CORAIL, c/o Institut Halieutique et des Sciences Marines (IH.SM), Universite de Toliara, Route du port, Toliara, P 141, 601 B, Madagascar 1 E-mail:rblob@clemson.edu Synopsis The evolution of novel functional traits can contribute substantially to the diversification of lineages. Older functional traits might show greater variation than more recently evolved novelties, due to the accrual of evolutionary changes through time. However, functional complexity and many-to-one mapping of structure to function could complicate such expectations. In this context, we compared kinematics and performance across juveniles from multiple species for two styles of waterfall-climbing that are novel to gobiid fishes: ancestral ‘‘powerburst’’ climbing, and more recently evolved ‘‘inching’’, which has been confirmed only among species of a single genus that is nested within the clade of powerburst climbers. Similar net climbing speeds across inching species seem, at first, to indicate that this more recently evolved mode of climbing exhibits less functional diversity. However, these similar net speeds arise through different pathways: Sicyopterus stimpsoni from Hawai’i move more slowly than S. lagocephalus from La Reunion, but may also spend more time moving. The production of similar performance between multiple functional pathways reflects a situation that resembles the phenomenon of many-to-one mapping of structure to function. Such similarity has the potential to mask appropriate interpretations of relative functional diversity between lineages, unless the mechanisms underlying performance are explored. More specifically, similarity in net performance between ‘‘powerburst’’ and ‘‘inching’’ styles indicates that selection on climbing performance was likely a limited factor in promoting the evolution of inching as a new mode of climbing. In this context, other processes (e.g., exaptation) might be implicated in the origin of this functional novelty. Synopsis FRENCH: Diversite fonctionnelle des innovations evolutives: l’exemple de la cinematique et des performances de grimpe des chutes d’eau des juveniles de gobies Resume L’evolution de nouveaux traits fonctionnels peut contribuer significativement a la diversification des lignees. Les traits fonctionnels les plus anciens peuvent montrer plus de variabilite que les plus recents du fait de l’accumulation de changements evolutif au cours du temps. Cependant, ces predictions peuvent ^etre complexifiees par la diversite des fonctions et par l’implication de plusieurs structures dans une m^eme fonction. Dans ce contexte, nous avons etudie la cinematique et les performances de grimpe des chutes d’eau de plusieurs especes de gobies utilisant deux styles de grimpe originaux au sein de cette famille: le mode « powerburst » plus ancestral et le mode « inching » qui a evolue plus recemment. Le mode inching n’a ete confirme que pour deux especes du m^eme genre incluses au sein du clade des powerburst. Des vitesses de grimpe similaires entre les especes utilisant le mode inching paraissent indiquer que ce mode de grimpe, qui a evolue plus recemment, presente une diversite fonctionnelle moins elevee. Toutefois, la similarite des vitesses de grimpe entre les deux especes s’explique par des processus differents: le Sicyopterus stimpsoni d’Haiwaı̈ se deplace plus lentement que le S. lagocephalus de La Reunion mais passe plus de temps en mouvement. La production de performances similaires, resultant de processus differents, reflete un phenomene semblable a celui de l’implication de plusieurs structures dans une m^eme fonction. Si les mecanismes sous-jacents ne sont pas explores, ces similarites peuvent perturber l’interpretation des differences relatives de diversite fonctionnelle entre les lignees. C The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. V 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. Downloaded from https://academic.oup.com/iob/article/1/1/obz029/5628227 by guest on 18 May 2024 R.W. Blob D. Ponton R.W. Blob et al. 2 Par ailleurs, les performances de grimpe similaires entre certaines especes utilisant le mode inching et d’autres le mode powerburst paraissent indiquer que la force de selection sur les performances de grimpe est sans doute un facteur reduisant l’avantage evolutif du mode de grimpe inching. Dans ce contexte, d’autres mecanismes (e.g., exaptation) pourraient ^etre a l’origine de cette innovation fonctionnelle. Translated to French by Raphael Lagarde (raph.lagarde@gmail. com) The evolution of novel functional abilities is widely regarded as a key factor that can influence the ecological diversification of lineages (Liem 1973; Wainwright 2007). Through the evolution of novel functional capacities, opportunities to exploit new resources or habitats may open, promoting diversification through either the advent of new specializations, or the radiation of taxa within a lineage (e.g., Konow et al. 2008). Alternatively, some functional novelties may have little impact on diversification (e.g., Price et al. 2010; Wainwright and Price 2016), or even reduce aspects of functional diversity in a lineage (e.g., Higham et al. 2015). Numerous factors might affect how novel traits contribute to ecological and functional diversification (Vermeij 2001). For example, older traits might show greater functional variation due to the accrual of evolutionary changes through time (Wainwright and Price 2016). Alternatively, novel functional capacities could emerge through evolutionary transitions that pass through a flexible, intermediate range of performance between lower and higher extremes (Blob 2001). In such cases, recently evolved novelties might show greater variation in performance than older traits. Beyond such considerations, the complexity of many functional systems (Wainwright 2007) leads to a potential for multiple structural arrangements of features to produce similar functional performance (many-to-one mapping of structure to function: Alfaro et al. 2005; Wainwright et al. 2005). Such circumstances could further complicate expectations for the functional diversity that would be exhibited by novel evolutionary traits. The waterfall-climbing abilities found in multiple species of gobiid fishes represent a remarkable functional novelty, through which potential patterns of diversification in functional performance can be tested. Waterfall-climbing is common among amphidromous juvenile gobies that are returning to adult stream habitats. After completing a marine larval phase, climbing is facilitated by a ventral sucker comprised of the fused pelvic fins (Schoenfuss and Blob 2003; Blob et al. 2006; Schoenfuss et al. 2011). Previous observations identified two distinct modes of climbing used by different species of gobies. ‘‘Powerburst’’ climbers use pectoral fin adduction and bursts of axial undulation to propel themselves upwards between periods of attachment to the substrate with the pelvic sucker. In contrast, ‘‘inching’’ climbers sequentially detach and reattach the pelvic sucker and a novel, oral sucker to climb, with little axial movement. Inching has been confirmed only in the genus Sicyopterus (Schoenfuss and Blob 2003; Blob et al. 2006; Schoenfuss et al. 2011; Maie et al. 2012), which is phylogenetically nested within several powerburst outgroups (Taillebois et al. 2014; Fig. 1). Thus, powerburst climbing appears to be ancestral, and inching more recently evolved (Cullen et al. 2013). Comparisons among powerburst species from Hawai’i and the Caribbean have shown a wide range of performance within this climbing mode. However, inching performance has only been measured in one species, S. stimpsoni from Hawai’i (Schoenfuss and Blob 2003; Blob et al. 2006; Schoenfuss et al. 2011). To evaluate whether more recently evolved novel traits, like inching, might show less functional diversity than ancestral traits, like powerburst climbing, we compared our previous data on climbing kinematics and velocity from Hawaiian and Caribbean species (Schoenfuss and Blob 2003; Blob et al. 2006; Schoenfuss et al. 2011) to new data from two additional species from the Indian Ocean island of La Reunion: the inching climber S. lagocephalus, and the powerburst climber Cotylopus acutipinnis. Materials and methods Experiments were conducted under St. Cloud State University Institutional Animal Care and Use Committee guidelines (protocol 12-07). Sampling in La Reunion was conducted under permit N 15002/DEAL/SEB/UPEMA issued by Direction de Downloaded from https://academic.oup.com/iob/article/1/1/obz029/5628227 by guest on 18 May 2024 Introduction 3 Functional novelties in gobiid fishes l’Environnement de l’Amenagement et du Logement de La Reunion. Juvenile S. lagocephalus and C. acutipinnis were collected by electroshocking from the St. Etienne River in La Reunion during April 2015. Fish were collected near the river mouth to ensure that they were recent, postmetamorphic recruits that had not previously climbed. Fish were transported by car to the Hydro Reunion laboratory facility, where they were maintained in aerated stream water at ambient temperature (20 C) and a 12:12 h cycle of room lighting. Fish were acclimated a minimum of 24 h prior to testing, which proceeded for 1–2 days after collection. Rocks were placed in enclosures to provide cover and a source of algal growth for food. To facilitate comparisons across species, kinematic and performance data from La Reunion gobies were collected and analyzed following methods of previous studies as closely as possible (Schoenfuss and Blob 2003; Blob et al. 2006; Schoenfuss et al. 2011). Briefly, the kinematics of individual climbing cycles were recorded in ventral view using highspeed video (100–200 Hz, Fastec Highspec 2G) as groups of fish ascended an inclined panel of Plexiglas (52 , lightly roughened with sandpaper) with water flowing as a sheet over its surface (180 mL s1). Lighting was provided by light-emitting diode (LED) fixtures or ambient room lighting. Measurements were performed by tracking landmarks on the head, fins, body axis and suckers (Fig. 2) using DLTdv5 for MATLAB (Hedrick Fig. 2 Still image of juvenile S. lagocephalus, extracted from highspeed video footage, illustrating the anatomical landmarks that were digitized for kinematic analyses. Because the fish is filmed in ventral view through Plexiglas, references will be made to anatomical left and right, which are opposite of what they appear in the image. (1) anterior midline edge of upper lip; (2) right edge of lip; (3) left edge of lip; (4) posterior midline of oral sucker (for S. lagocephalus) or head (for C. acutipinnis); (5) base of right pectoral fin; (6) tip of right pectoral fin; (7) base of the left pectoral fin; (8) tip of the left pectoral fin; (9) anterior edge of pelvic sucker; (10–16) evenly spaced body axis points; (17) caudal peduncle. 2008), and using the coordinate data for these landmarks to calculate kinematic variables in MATLAB (Schoenfuss and Blob 2003; Schoenfuss et al. 2011). Performance trials were recorded in dorsal view with Sony Handycam mini-DV cameras (60 Hz), over a standard distance of 20 cm (10 body lengths), as fish ascended an inclined (70 ), textured plastic chute with water flowing down its surface (Blob et al. 2006). Video records were used to determine the portion of time each fish spent moving versus resting as it ascended the 20 cm distance, from which we calculated climbing speeds while moving and net climbing speeds including rest time. Performance metrics were compared across species via Kruskal–Wallis analyses with Dunn’s post-hoc tests corrected for multiple comparisons, using the Prism 6.1 statistical package (Graph-Pad software). Results Inching S. lagocephalus from La Reunion showed similar kinematic profiles to patterns observed previously in S. stimpsoni from Hawai’i (Fig. 3A; Supplementary Movies S1–S2). The area of the oral sucker initially decreases as the front lip advances Downloaded from https://academic.oup.com/iob/article/1/1/obz029/5628227 by guest on 18 May 2024 Fig. 1 Phylogenetic relationships of gobiid species from which climbing performance has been evaluated, based on previous published analyses (Taillebois et al. 2014). Geographic location of each taxon is indicated in parentheses after its name: HI, Hawai’i; REU, La Reunion; CARIB, Caribbean (Dominica). Modes of climbing are indicated to the right of species names. Stenogobius hawaiiensis is included for reference as a non-climbing outgroup taxon. Dashed line indicates differing phylogenetic relationships of the genus Awaous. Based on available data, inching evolved once within the genus Sicyopterus. 4 R.W. Blob et al. Downloaded from https://academic.oup.com/iob/article/1/1/obz029/5628227 by guest on 18 May 2024 Fig. 3 Comparative kinematics of juvenile waterfall-climbing gobies. (A) Inching climbers. Mean profiles for kinematic variables during inching climbing by S. stimpsoni from Hawai’i (n ¼ 26 climbing cycles, left column; data from Schoenfuss and Blob 2003) and S. lagocephalus from La Reunion: (n ¼ 86 climbing cycles, right column). All trials were normalized to the same time duration, and plots show mean ± SE for every 2% increment of locomotor cycle duration. (B) Powerburst climbers. Mean profiles for axial kinematics during vertical powerburst climbing by juvenile A. stamineus and L. concolor from Hawai’i (n ¼ 17 climbing cycles, left column; data pooled for these species as reported by Schoenfuss and Blob 2003), S. punctatum from Dominica (n ¼ 22 climbing cycles, middle column; data from Schoenfuss et al. 2011); and C. acutipinnis from La Reunion (n ¼ 87 climbing cycles, right column). Top row: bars for each equal-length segment of the body plot the mean (± SE) maximum angle of that segment to the direction of travel at any point during the cycle. Bottom row: mean (± SE) maximum amplitudes throughout the climbing cycle for each of 11 equally spaced points along the length of the fish, normalized as a percentage of body length. Original data reported in Supplementary Table S1. 5 Functional novelties in gobiid fishes Table 1. Means (± SD) of morphological and performance variables for juveniles of waterfall climbing stream gobies from Hawai’i (A. stamineus, L. concolor, S. stimpsoni), Dominica (S. punctatum) and La Reunion (C. acutipinnis, S. lagocephalus), with numbers in parentheses indicating the sample size of locomotor events from which data were collected with each cycle from a different individual Hawai’i Variable Fish length (cm)1 Climbing bout duration (s)1,2 Awaous stamineus 1.4 (12) 1.4 (17) 1.81±0.85a(12) 1.97±0.7a(17) abc Net climbing speed including 0.21±0.10 rest (cm/s)1,2 Sicyopterus stimpsoni 2.2 (17) Sicydium punctatum 1.9 (39) La R eunion Cotylopus acutipinnis Sicyopterus lagocephalus 1.6±0.5 (11) 2.5±0.4 (62) Kruskal– Wallis P-value n/a 7.55±6.5b(17) 2.51±1.85a(39) 2.49±0.52ac(11) 3.34±1.51c(62) <0.001 (12) 0.21±0.09 (17) 0.22±0.07b(17) 0.21±0.13ab(39) 0.09±0.03c(11) 0.28±0.11ab(62) <0.001 b 0.15±0.07a(12) 0.15±0.06a(17) 0.10±0.03ab(17) 0.11±0.07ab(39) 0.06±0.03b(11) 0.11±0.05a(62) <0.001 Speed during climbing only (cm/s)1,2 1.17±0.46ab(12) 0.94±0.14a(17) 0.34±0.07bc(17) 1.02±0.47a(39) 0.73±0.25a(11) 0.62±0.12c(62) <0.001 Speed during climbing only (BL/s)1,2 0.84±0.33a(12) 0.67±0.10a(17) 0.15±0.03b(17) 0.54±0.25a(39) 0.50±0.23a(11) 0.25±0.07c(62) <0.001 Percent time in motion1–3 20.0±10.3a(12) 22.3±11a(17) 54.3±21b(16) 21.6±11.6a(39) 13.5±5.4a(11) 47.2±18.4b(62) <0.001 1 Data for Hawaiian species from (Blob et al. 2006), data for S. punctatum from (Schoenfuss et al. 2011), data for species from La Reunion new in this study. 2 Superscript letters (a, b, c) indicate groupings of significantly different species (P < 0.05), based on Dunn’s post-hoc tests corrected for multiple comparisons, conducted after Kruskal–Wallis analyses. 3 Data arcsine transformed prior to Kruskal–Wallis analysis. n/a, not applicable; BL/s, body lengths per second. early in the cycle. However, the area of the oral sucker then increases as fish reattach it to the substrate while the pelvic sucker advances up the surface. Ranges of values for kinematic variables are also similar between the species, with the slightly longer upward advance of the oral and pelvic suckers during each cycle in S. lagocephalus corresponding to the slightly larger average body size of this species (25 mm standard length) compared to S. stimpsoni (22 mm; Table 1; Schoenfuss and Blob 2003). Kinematic profiles across powerburst climbers also show generally similar patterns, with moderate ranges of variation in kinematic variables (Fig. 3B; Supplementary Movies S3–S4). In Hawaiian powerburst climbers, Sicydium punctatum from the Caribbean and C. acutipinnis from La Reunion, body segments near the head and tail show the greatest amplitudes during axial undulation, with caudal body segments showing the greatest angles to the direction of travel (Fig. 3B). However, larger C. acutipinnis (16 mm) and S. punctatum (19 mm) exhibit larger values for both of these variables than the smaller (14 mm) Hawaiian taxa. In contrast to kinematic comparisons, the two climbing styles show different patterns of variation in performance across taxa. With regard to net climbing speed over 20 cm, including periods of motion and rest, the two species of inching climbers show similar performance to each other (and to three of the four powerburst climbers: mean 0.10– 0.14 BL s1; Table 1; Fig. 4A). In contrast, the powerburst climber C. acutipinnis from La Reunion (mean 0.06 BL s1) is significantly slower than two of the other powerburst climbers (Awaous stamineus and Lentipes concolor), as well as one of the inching species (S. lagocephalus) (Dunn’s post-hoc test P < 0.05; Table 1; Fig. 4A). Thus, powerburst climbers include species with at least two distinct levels of net climbing performance, whereas inching climbers include only one such group. Across all of the variables that we compared, the average coefficient of variation was also greater for powerburst climbing species (45 ± 3.1 standard error [SE]) than for inching species (33 ± 3.2 SE) (Mann–Whitney Utest, P < 0.01). However, this apparent greater performance variation among powerburst climbers is a product of the variables that underlie this performance. Comparisons of movement speed (i.e., speed restricted to periods of actual movement) show significant differences between the two inching climbers, whereas the powerburst climbers group similarly (Table 1; Fig. 4B). It is noteworthy, though, that C. acutipinnis uses the slowest movement speeds among powerburst species (Table 1; Fig. 4B). Comparisons of the time spent in motion over 20 cm show distinctions between the powerburst (<25%) versus inching (50%) climbers (P < 0.05), with species within each climbing style Downloaded from https://academic.oup.com/iob/article/1/1/obz029/5628227 by guest on 18 May 2024 Net climbing speed including rest (BL/s)1,2 Lentipes concolor Dominica R.W. Blob et al. 6 Discussion Recently evolved novelties have had less time to accrue evolutionary changes that might contribute to functional diversity than older traits. As a result, older traits might be expected to show more variation than recent evolutionary novelties (Wainwright and Price 2016). However, our results from climbing gobies illustrate how the equivalent performance of alternative functional pathways can influence interpretations of the relative diversity exhibited by functional traits. Comparisons of net climbing speed between inching and powerburst species gave an initial indication that more recently evolved climbing by inching was largely similar in performance across species, whereas the older trait of powerburst climbing displayed greater variation across the taxa that retain this climbing mode (Table 1; Fig. 4A). In contrast, comparisons of the mechanisms that underlie the performance of both styles present a different picture, in which powerburst climbing species might show more consistent average patterns than inchers (Fig. 4B). These patterns result because, within each climbing style, species appear to use largely similar kinematics in different ways—for example, by spending more (or less) time moving compared to resting (Fig. 4C). Thus, without considering the mechanics that underlie performance, the functional diversity Fig. 4 Comparative climbing performance of juvenile waterfallclimbing gobies, measured over 20 cm distances. (A) Net speed including periods of rest, normalized for body size. (B) Speed during periods of movement only, normalized for body size. (C) Proportion of time spent moving during 20 cm trials. Boxes show 25th percentile, median, and 75th values; whiskers illustrate 10th and 90th percentile values; open circles indicate values outside these percentiles. Vertical dashed line in each plot divides species by climbing style (powerburst climbers on the left, inching climbers on the right). Colors represent differences in locality, with white boxes showing data from Hawaiian species derived from Blob et al. (2006), gray boxes data from Caribbean species derived from Schoenfuss et al. (2011), and orange boxes new data from Reunionese species. Different boldface letters above each box plot indicate significant differences between groups, determined by Kruskal–Wallis analyses with Dunn’s posthoc tests, corrected for multiple comparisons (P < 0.05). As, A. stamineus; Lc, L. concolor; Sp, S. punctatum; Ca, C. acutipinnis; Ss, S. stimpsoni; Sl, S. lagocephalus. Original data and sample sizes are reported in Supplementary Table S2. Downloaded from https://academic.oup.com/iob/article/1/1/obz029/5628227 by guest on 18 May 2024 grouping together (Table 1; Fig. 4C). However, both C. acutipinnis and S. lagocephalus from La Reunion showed lower mean values than other species using their respective climbing styles. As a result, the similar net speeds of inching climbers seem to arise through different pathways: S. stimpsoni from Hawai’i move more slowly than S. lagocephalus from La Reunion, but may spend more time moving. In contrast, the cumulative effect of seemingly minor differences in both movement speed and the portion of time spent moving, differences that were not initially recognized as significant, led to distinctly slow net speeds for C. acutipinnis among powerburst species. 7 Functional novelties in gobiid fishes (Teichert et al. 2014). Hawaiian species of amphidromous gobies show even more limited correlations between climbing performance and in-stream distribution. Although the two Hawaiian powerburst species exhibit nearly identical net speeds, one species (A. stamineus) penetrates the shortest distance upstream and the other species (L. concolor) penetrates the furthest, with inching S. stimpsoni reaching intermediate distances between the two (Schoenfuss and Blob 2003, 2007). Given that climbing performance does not predict ecological factors such as habitat distribution, changes in net performance appear unlikely to have formed a selective basis for the evolutionary origin of inching as a functional novelty in climbing style. Instead, the origin of inching with an oral sucker may have been tied to non-locomotor pressures, potentially reflecting an exaptation of feeding adaptations for locomotor roles (Cullen et al. 2013). Authors’ contributions R.W.B., R.L., and H.L.S. designed the study and collected data. All authors contributed to data analysis, interpretation, and manuscript development, and approved the final manuscript. Acknowledgements We thank H. Grondin, G. Borie, and T. Offerle for contributions to data collection and analysis. Funding This work was supported by the National Science Foundation [DBI-146089] and a Saint Cloud State University Scholar award [211550 to H.L.S.]. Supplementary data Supplementary data available at IOB online. References Alfaro ME, Bolnick DI, Wainwright PC. 2005. Evolutionary consequences of many-to-one mapping of jaw morphology to mechanics in labrid fishes. Am Nat 165:E140–54. Blob RW. 2001. Evolution of hindlimb posture in nonmammalian therapsids: biomechanical tests of paleontological hypotheses. Paleobiology 27:14–38. Blob RW, Rai R, Julius ML, Schoenfuss HL. 2006. Functional diversity in extreme environments: effects of locomotor style and substrate texture on the waterfall climbing performance of Hawaiian gobiid fishes. J Zool 268:315–24. Cullen JA, Maie T, Schoenfuss HL, Blob RW. 2013. Evolutionary novelty versus exaptation: oral kinematics in feeding versus climbing in the waterfall-climbing Hawaiian goby Sicyopterus stimpsoni. PLoS One 8:e53274. Downloaded from https://academic.oup.com/iob/article/1/1/obz029/5628227 by guest on 18 May 2024 present within more recently evolved inching might have been masked. Such masking could lead to a potentially oversimplified conclusion that functional diversity is lower in the more recent evolutionary novelty. The complexity of many functional systems opens opportunities for similar performance to be produced through multiple pathways. One variety of this general phenomenon that has received considerable attention is the many-to-one mapping of structure to function, in which multiple anatomical arrangements of structures may produce equivalent functional performance (Alfaro et al. 2005; Wainwright et al. 2005). Our data from waterfallclimbing gobies provide an example of the broader phenomenon of which many-to-one mapping of structure to function is a part, in which alternative functional pathways—such as different climbing styles, or different patterns of motion versus rest within a climbing style—can produce equivalent locomotor performance. For example, three of the four powerburst species compared show net climbing speeds that can be statistically grouped with those of both inching species observed, despite using different propulsive mechanisms to scale waterfalls (Table 1; Fig. 4A). In addition, among inching species, equivalent climbing performance can be achieved by longer durations of slow movement (e.g., S. stimpsoni) and shorter durations of faster movement (e.g., S. lagocephalus; Table 1; Fig. 4). The ultimate similarity of net climbing performance between the two broad styles of climbing in gobies is striking. Out of the six species we compared, from islands in three different ocean basins (Pacific, Caribbean, and Indian), five showed net climbing speeds between 0.10 and 0.14 BL s1. Only C. acutipinnis used markedly slower speeds (0.06 BL s1), which were a product of moderately slower movement and a smaller proportion of time spent moving, but which still overlapped with the net speeds of some of the other five species (Fig. 4). Given that climbing behaviors are components of a migratory phase of the life cycle, to what extent might differences in performance impact features such as the in-stream distributions and upstream penetration of species? In La Reunion, C. acutipinnis has a higher failure rate during attempted climbs than faster S. lagocephalus (Lagarde et al. 2018), and it takes C. acutipinnis 20–25% more days to migrate to adult upstream habitats (Lagarde et al. 2020). Although this could allow faster juvenile S. lagocephalus to reach suitable habitat before C. acutipinnis, both species ultimately penetrate to comparable upstream distances 8 Schoenfuss HL, Blob RW. 2007. The importance of functional morphology for fishery conservation and management: applications to Hawaiian amphidromous fishes. Bishop Mus Bull Cult Environ Stud 3:125–141. Schoenfuss HL, Maie T, Kawano SM, Blob RW. 2011. Performance across extreme environments: comparing waterfall climbing among amphidromous gobioid fishes from Caribbean and Pacific islands. Cybium 35:361–9. Taillebois L, Castelin M, Lord C, Chabarria R, Dettaı̈ A, Keith P. 2014. New Sicydiinae phylogeny (Teleostei: Gobioidei) inferred from mitochondrial and nuclear genes: insights on systematics and ancestral areas. Mol Phylog Evol 70: 260–71. Teichert N, Valade P, Lim P, Dauba F, Labonne J, Richarson M, Bosc P, Gaudin P. 2014. Habitat selection in amphidromous Gobiidae of Reunion Island: Sicyopterus lagocephalus (Pallas, 1770) and Cotylopus acutipinnis (Guichenot, 1863). Environ Biol Fishes 97:255–66. Vermeij GJ. 2001. Innovation and evolution at the edge: origins and fates of gastropods with a labral tooth. Biol J Linn Soc Lond 72:461–508. Wainwright PC. 2007. Functional versus morphological diversity in macroevolution. Ann Rev Ecol Evol Syst 38:381–401. Wainwright PC, Alfaro ME, Bolnick DI, Hulsey CD. 2005. Many-to-one mapping of form to function: a general principle in organismal design? Integr Comp Biol 45:256–62. Wainwright PC, Price SA. 2016. The impact of organismal innovation on functional and ecological diversification. Integr Comp Biol 56:479–88. Downloaded from https://academic.oup.com/iob/article/1/1/obz029/5628227 by guest on 18 May 2024 Hedrick TL. 2008. Software techniques for two- and threedimensional kinematic measurements of biological and biomimetic systems. Bioinsp Biomim 3:034001. Higham TE, Birn-Jeffery AV, Collins CE, Hulsey CD, Russell AP. 2015. Adaptive simplification and the evolution of gecko locomotion: morphological and biomechanical consequences of losing adhesion. Proc Natl Acad Sci U S A 112:809–14. Konow N, Bellwood DR, Wainwright PC, Kerr AM. 2008. Evolution of novel jaw joints promote trophic diversity in coral reef fishes. Biol J Linn Soc Lond 93:545–55. Lagarde R, Borie G, Blob RW, Schoenfuss HL, Ponton D. 2018. Intra- and inter-specific morphological diversity of amphidromous gobies influences waterfall-climbing performance. J Zool 306:243–51. Lagarde R, Borie G, Ponton D. 2020. Dams select individual morphology but do not modify upstream migration speed of tropical amphidromous gobies. River Res Appl 36:57–67. Liem KF. 1973. Evolutionary strategies and morphological innovations: cichlid pharyngeal jaws. Syst Biol 22:425–41. Maie T, Schoenfuss HL, Blob RW. 2012. Performance and scaling of a novel locomotor structure: adhesive capacity of climbing gobiid fishes. J Exp Biol 215:3925–36. Price SA, Wainwright PC, Bellwood DR, Kazancioglu E, Collar DC, Near TJ. 2010. Functional innovations and morphological diversification in parrotfishes. Evolution 64:3057–68. Schoenfuss HL, Blob RW. 2003. Kinematics of waterfall climbing in Hawaiian freshwater fishes (Gobiidae): vertical propulsion at the aquatic-terrestrial interface. J Zool 261:191–205. R.W. Blob et al. 9 Functional novelties in gobiid fishes Synopsis GERMAN: Die Funktionelle Leistung von Evolution€aren Neuheiten: Erkenntnisse durch Kinematische- und Leistungsstudien von Wasserfallklettern an jugendlichen Gobiid-Fischen Zusammenfassung Die Entwicklung neuartiger funktioneller Merkmale kann wesentlich zur Diversifizierung von Abstammungslinien bei€ tragen. Altere funktionelle Merkmale k€ onnen aufgrund der im Laufe der Zeit auftretenden evolution€aren Ver€anderungen gr€ oßere Unterschiede aufweisen als neuere Merkmale. Die funktionale Komplexit€at und die Eins-zu-Eins-Zuordnung von Struktur-zu-Funktion k€ onnen solche Erwartungen jedoch erschweren. In diesem Zusammenhang haben wir die Kinematik und Leistung von Jungtieren verschiedener Spezien von Gobiid-Fischen f€ ur zwei Arten des Wasserfallkletterns verglichen: das €altere ,,Powerburst ‘‘-Klettern und das in j€ ungster Zeit entwickelte ,,Inching‘‘, das nur in einer Gattung von Gobiid- Fischen vorkommt, die in der Gruppe der Powerburst-Kletterer eingeschlossen € ist. Ahnliche Netto-Klettergeschwindigkeiten bei ‘‘Inching’’Spezien scheinen zun€achst darauf hinzudeuten, dass diese neuere Art des Kletterns wenig funktionelle Vielfalt aufweist. Allerdings ist die €ahnliche Nettogeschwindigkeit das Resultat von verschiedenen Faktoren: Sicyopterus stimpsoni aus Hawaii bewegt sich langsamer als S. lagocephalus aus La Reunion aber bewegt sich mehr kontinuierlich. Das Ergebnis einer €ahnlichen Leistung durch zweierlei Funktionspfade €ahnelt dem Ph€anomen der Eins-zu-Eins Zuordnung von Struktur€ zu-Funktion. Eine solche Ahnlichkeit kann die angemessene Interpretation der relativen funktionalen Vielfalt zwischen den Linien erschweren, sofern nicht die der Leistung zugrundeliegenden Mechanismen untersucht werden. Insbesondere € zeigt die Ahnlichkeit in der Nettoleistung zwischen ‘‘Powerburst’’ - und ‘‘Inching’’ -Kletterstilen, dass die Entwicklung des neuen Kletterstils wahrscheinlich wenig mit verbesserter Kletterleistung zu tun hat. In diesem Zusammenhang k€ onnten andere Prozesse (z. B. Exaptation) an der Entstehung dieser funktionellen Neuheit beteiligt sein. Translated to German by Heiko Schoenfuss (hschoenfuss@stcloudstate.edu) PORTUGESE: Diversidade funcional de novidades evolucionarias: percepc~ oes da cinematica da escalada em cascatas e desempenho de peixes juvenis gobiıdeos Resumo A evoluc~ao de novos tracos funcionais pode contribuir substancialmente para a diversificac~ao de linhagens. Os tracos funcionais mais antigos podem mostrar maior variac~ao do que as novidades desenvolvidas mais recentemente, devido ao ac umulo de mudancas evolutivas ao longo do tempo. No entanto, a complexidade funcional e os in umeros mapeamentos de uma estrutura para uma nica func~ao podem complicar essas expectativas. Nesse u contexto, comparamos a cinematica e a performance em juvenis de varias especies para dois estilos de escalada em cascata que s~ao novidades em peixes gobiıdeos: a ancestral escalada por ‘‘explos~ao’’ e o evolutivamente mais recente chamado de ‘‘avancamento’’, que foi confirmado apenas nico g^enero que dentro do clado entre especies de um u de escaladores por explos~ao. Velocidades finais de escalada semelhantes entre especies usando ‘‘avancamento’’ parecem, inicialmente, indicar que esse modo de escalada evolutivamente mais recente exibe menor diversidade funcional. No entanto, essas velocidades finais similares ocorrem por diferentes formas: Sicyopterus stimpsoni do Havaı se move mais devagar que S. lagocephalus das Ilhas Reuni~ao, mas pode gastar mais tempo se movendo. Desempenhos semelhantes entre varias vias funcionais refletem uma situac~ao que se assemelha ao fen^ omeno de mapeamento de nica func~ao. Essa semelhanca tem uma estrutura para um u o potencial de ocultar interpretac~ oes apropriadas sobre relativa diversidade funcional entre linhagens, a menos que os mecanismos que afetam o desempenho sejam explorados. Mais especificamente, a semelhanca no desempenho final entre os estilos ‘‘explos~ao’’ e ‘‘avancamento’’indica que a selec~ao na performance em escalada provavelmente foi um fator limitante na promoc~ao da evoluc~ao por ‘‘avancamento’’ como um novo modo de escalada. Nesse contexto, outros processos (e.g., exaptac~ao) podem estar relacionados com a origem dessa novidade funcional. Translated to Portuguese by Diego Vaz (dbistonvaz@vims. edu) Downloaded from https://academic.oup.com/iob/article/1/1/obz029/5628227 by guest on 18 May 2024 Synopsis 10 R.W. Blob et al. Synopsis Downloaded from https://academic.oup.com/iob/article/1/1/obz029/5628227 by guest on 18 May 2024 JAPANESE: 進化的ノベルティの機能的多様性：滝登りハゼの稚魚によ る岩登り時の運動学とパフォーマンスからの洞察 （Abstract: 梗概） 進化的に新しい機能特性は系統の多様 化に大きく貢献することが可能である。より古い機能特性 は、時間の経過に伴う進化的変化の蓄積により、より新しく 発現した特性よりも大きな偏差を示すと考えられるのである が、機能の複雑さと、構造と機能における多対一の関連性 により、この様な予測はより複雑になる可能性がある。これを 踏まえて、我々は祖先的岩登りスタイルの‘‘パワーバースト’’タイ プとその系統群の中から進化的に新しく発現した単一の属が 獲得したとされる‘‘インチング’’タイプを示す複数種の滝登りハ ゼの稚魚による岩登り時の運動学とパフォーマンス比較分析し た。‘‘インチング’’タイプの実質的なクライミング速度はどれも 種間の差異が認められなかった、そのためこのもっとも最近進 化的に発現した岩登りのスタイルは、機能的多様性をあまり示 さないことを示唆しているよう考えられる。しかし、ハワイ島の Sicyopterus stimpsoniは、レユニオン島のS. lagocephalusよりも 遅いが、クライミング移動により多くの時間を費やす傾向にあ ることが分かった。したがって、この種間に差異のない実質的 速度は異なる経路を介して発生していると考えられ、複数の機 能経路間で同様のパフォーマンスが得られたことは、多対一 の関連性があると言える。そしてこれはパフォーマンスの基礎と なるメカニズムが明確にされない限り、系統間の相対的な機 能的多様性の適切な解釈を複雑にするであろう。より具体 的に言うなれば、‘‘パワーバースト’’タイプと‘‘インチング’’タイプ の実質的パフォーマンスの類似性は、クライミングパフォーマン スのへ淘汰が進化的に新しい岩登りスタイルとしての‘‘インチン グ’’タイプを確立する上で制限要因になり得る可能性が高い ことを示している。さらには、他のプロセス（例えば、外適 応）がこの機能的ノベルティの起源に関係している可能性が あると考えられる。 Translated to Japanese by Takashi Maie (maie.t@lynchburg.edu) 

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Atmos. Meas. Tech., 13, 3119–3146, 2020 https://doi.org/10.5194/amt-13-3119-2020 © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License. Implementation of a chemical background method for atmospheric OH measurements by laser-induced fluorescence: characterisation and observations from the UK and China Robert Woodward-Massey1,a , Eloise J. Slater1 , Jake Alen1 , Trevor Ingham1,2 , Danny R. Cryer1 , Leanne M. Stimpson1 , Chunxiang Ye1,a , Paul W. Seakins1 , Lisa K. Whalley1,2 , and Dwayne E. Heard1 1 School of Chemistry, University of Leeds, Leeds, LS2 9JT, UK Centre for Atmospheric Science, University of Leeds, Leeds, LS2 9JT, UK a now at: College of Environmental Sciences and Engineering, Peking University, Beijing, 100871, China 2 National Correspondence: Lisa K. Whalley (l.k.whalley@leeds.ac.uk) and Dwayne E. Heard (d.e.heard@leeds.ac.uk) Received: 17 December 2019 – Discussion started: 15 January 2020 Revised: 7 April 2020 – Accepted: 29 April 2020 – Published: 12 June 2020 Abstract. Hydroxyl (OH) and hydroperoxy (HO2 ) radicals are central to the understanding of atmospheric chemistry. Owing to their short lifetimes, these species are frequently used to test the accuracy of model predictions and their underlying chemical mechanisms. In forested environments, laser-induced fluorescence–fluorescence assay by gas expansion (LIF–FAGE) measurements of OH have often shown substantial disagreement with model predictions, suggesting the presence of unknown OH sources in such environments. However, it is also possible that the measurements have been affected by instrumental artefacts, due to the presence of interfering species that cannot be discriminated using the traditional method of obtaining background signals via modulation of the laser excitation wavelength (“OHwave ”). The interference hypothesis can be tested by using an alternative method to determine the OH background signal, via the addition of a chemical scavenger prior to sampling of ambient air (“OHchem ”). In this work, the Leeds FAGE instrument was modified to include such a system to facilitate measurements of OHchem , in which propane was used to selectively remove OH from ambient air using an inlet pre-injector (IPI). The IPI system was characterised in detail, and it was found that the system did not reduce the instrument sensitivity towards OH (< 5 % difference to conventional sampling) and was able to efficiently scavenge external OH (> 99 %) without the removal of OH formed inside the fluorescence cell (< 5 %). Tests of the photolytic interference from ozone in the presence of water vapour revealed a small but potentially significant interference, equivalent to an OH concen- tration of ∼ 4 × 105 molec. cm−3 under typical atmospheric conditions of [O3 ] = 50 ppbv and [H2 O] = 1 %. Laboratory experiments to investigate potential interferences from products of isoprene ozonolysis did result in interference signals, but these were negligible when extrapolated down to ambient ozone and isoprene levels. The interference from NO3 radicals was also tested but was found to be insignificant in our system. The Leeds IPI module was deployed during three separate field intensives that took place in summer at a coastal site in the UK and both in summer and winter in the megacity of Beijing, China, allowing for investigations of ambient OH interferences under a wide range of chemical and meteorological conditions. Comparisons of ambient OHchem measurements to the traditional OHwave method showed excellent agreement, with OHwave vs OHchem slopes of 1.05–1.16 and identical behaviour on a diel basis, consistent with laboratory interference tests. The difference between OHwave and OHchem (“OHint ”) was found to scale nonlinearly with OHchem , resulting in an upper limit interference of (5.0 ± 1.4) ×106 molec. cm−3 at the very highest OHchem concentrations measured (23×106 molec. cm−3 ), accounting for ∼ 14 %–21 % of the total OHwave signal. Published by Copernicus Publications on behalf of the European Geosciences Union. 3120 1 R. Woodward-Massey et al.: Implementation of a chemical background method Introduction The removal of pollutants and greenhouse gases in the troposphere is dominated by reactions with the hydroxyl radical (OH), which is closely coupled to the hydroperoxy radical (HO2 ). Comparisons of the levels of OH and HO2 observed during field campaigns to the results of detailed chemical box models serve as a vital tool to assess our understanding of the underlying chemical mechanisms involved in tropospheric oxidation. Laser-induced fluorescence–fluorescence assay by gas expansion (LIF–FAGE) measurements of OH in forested environments have often been considerably higher than those predicted by models (Carslaw et al., 2001; Lelieveld et al., 2008; Ren et al., 2008; Hofzumahaus et al., 2009; Stone et al., 2011; Whalley et al., 2011; Wolfe et al., 2011). The difficulty in simulating radical concentrations in such environments has prompted a multitude of theoretical (Peeters et al., 2009, 2014; da Silva et al., 2010; Nguyen et al., 2010; Peeters and Muller, 2010), laboratory (Dillon and Crowley, 2008; Hansen et al., 2017), and chamber (Paulot et al., 2009; Crounse et al., 2011, 2012; Wolfe et al., 2012; Fuchs et al., 2013, 2014, 2018; Praske et al., 2015; Teng et al., 2017) studies to help explain the sources of the measurement–model discrepancy through detailed investigations of the mechanism of isoprene oxidation under low NOx conditions, as well as other biogenic volatile organic compounds (BVOCs) (Novelli et al., 2018). However, another hypothesis is that the LIF measurements have, at least in part, suffered from an instrumental bias in these environments due to interfering species. Early LIF measurements of OH suffered from significant interferences due to laser-generated OH from ozone photolysis in the presence of water vapour (Hard et al., 1984). While this effect has been reduced by going from 282 to 308 nm laser excitation of OH, it may still be significant, especially at night or with the use of multi-pass laser set-ups (e.g. up to ∼ 4 × 106 molec. cm−3 in Griffith et al., 2016). Laboratory experiments conducted by Ren and co-workers using the Pennsylvania State University (PSU) LIF instrument showed negligible interferences in OH detection for a range of candidate species: H2 O2 , HONO, HCHO, HNO3 , acetone, and various RO2 radicals (Ren et al., 2004). Observations of OH during the PROPHET (Program for Research on Oxidants: PHotochemistry, Emissions and Transport) field campaign in summer 1998, located in a mixed deciduous forest in Michigan, USA, revealed unusually high night-time OH concentrations (∼ 1×106 molec. cm−3 ) but measurement interferences were ruled out (Faloona et al., 2001). However, the results of more recent studies conducted in forested environments have meant that interferences in the measurement of OH by LIF–FAGE have been revisited. The usual background method of this technique, where the laser wavelength is scanned off-resonance from an OH transition (“OHwave ”), does not discriminate between ambient (i.e. “real”) OH and either OH formed inside the FAGE cell Atmos. Meas. Tech., 13, 3119–3146, 2020 (e.g. laser- or surface-generated OH or via unimolecular decomposition in the gas phase to form OH) or fluorescence from other species at λ ∼ 308 nm (e.g. naphthalene, SO2 ), although it is possible to correct for such effects providing the interference has been previously characterised (Martinez et al., 2004; Ren et al., 2004; Griffith et al., 2013; Fuchs et al., 2016). There is an alternative, chemical method (“OHchem ”) for obtaining the OH background signal in LIF instruments that allows for interference signals to be determined without their prior characterisation, in order to rule out possible artefacts from unknown species. The OHchem method involves the addition of a high concentration of an OH scavenger, such as perfluoropropene (C3 F6 ) or propane, just before the FAGE inlet. Ambient OH is quickly titrated away by fast reaction with the scavenger, but any interference should remain in the fluorescence signal, although this must be corrected for reaction of any internally generated OH with the scavenger inside the FAGE cell. Several LIF–FAGE groups have now made efforts to validate ambient OH measurements through incorporation of the alternative OHchem technique, which was first applied for continuous OH measurements by Mao et al. (2012). Since then, field studies of OH measurement interferences have been conducted in forested (Griffith et al., 2013; Novelli et al., 2014a, 2017; Feiner et al., 2016; Lew et al., 2019), rural (Fuchs et al., 2017; Tan et al., 2017), suburban (Tan et al., 2018, 2019), urban (Ren et al., 2013; Brune et al., 2016; Griffith et al., 2016), and coastal (Novelli et al., 2014a; Mallik et al., 2018) locations. Substantial improvement in measurement–model agreement has been possible when OH backgrounds were determined chemically, especially in forested environments, suggesting that understanding of tropospheric oxidation processes in such regions may be better than previously thought (Mao et al., 2012; Hens et al., 2014; Feiner et al., 2016). This is further supported by the positive identification of two new OH interference candidates in laboratory experiments, namely intermediates in alkene ozonolysis reactions, which may (Novelli et al., 2014b, 2017; Rickly and Stevens, 2018) or may not (Fuchs et al., 2016) be related to stabilised Criegee intermediates (SCIs), and the NO3 radical (Fuchs et al., 2016), although for all cases the observed interferences cannot explain the magnitudes of the OH background signals under ambient conditions. The trioxide species, ROOOH, has also been postulated to explain elevated OH backgrounds in LIF–FAGE measurements made in forested regions (Fittschen et al., 2019). However, it is not known whether other LIF instruments suffer the same levels of interference, which are likely highly dependent on cell design and operating parameters, especially the residence time of air between sampling and detection (Novelli et al., 2014a; Fuchs et al., 2016; Rickly and Stevens, 2018). Considering the bespoke nature of LIF– FAGE instruments, those of different groups share the same main features but differ in many aspects, such as inlet size and shape, or whether the laser crosses the detection axis https://doi.org/10.5194/amt-13-3119-2020 R. Woodward-Massey et al.: Implementation of a chemical background method once (i.e. single-pass) or multiple times (multi-pass). As a result, the magnitude of any interference is likely to vary significantly between different instruments. Because of this, a general recommendation of the 2015 International HOx Workshop (Hofzumahaus and Heard, 2016) was that different groups should incorporate their own chemical scavenger system for use in ambient OH measurements and to test interferences in the laboratory. Following this recommendation, the Leeds ground-based FAGE instrument was modified to incorporate a chemical scavenger system, through the addition of an inlet preinjector (IPI). In this work, we describe the design of the IPI system and its thorough characterisation in terms of sensitivity and the degree of external and internal OH removal. Following this, we present the results of interference testing experiments performed using the IPI system, in which we investigated interferences from O3 +H2 O, isoprene ozonolysis, and NO3 radicals. Finally, we demonstrate the use of the optimised IPI system for measurements of ambient OH made during three separate field campaigns in the UK and China, which encompassed a wide range of chemical and meteorological conditions. 2 2.1 Methodology Overview of the Leeds ground-based FAGE instrument The University of Leeds ground-based FAGE instrument, described in detail elsewhere (Creasey et al., 1997a; Whalley et al., 2010, 2013), has participated in 24 intensive field campaigns since its initial deployment in 1996. Measurements of OH, HO2 , and, more recently, RO2 radicals (Whalley et al., 2013) have been made in a variety of locations, ranging from pristine marine boundary layer (Creasey et al., 2003; Whalley et al., 2010), tropical rainforest (Whalley et al., 2011), and polar (Bloss et al., 2007) environments to coastal (Smith et al., 2006) and semi-polluted regions (Creasey et al., 2001), as well as urban areas (Heard et al., 2004; Emmerson et al., 2007), including a highly polluted megacity (Lee et al., 2016; Whalley et al., 2016). Ambient OH is measured using laser-induced fluorescence. Briefly, ambient air is drawn through a 1.0 mm diameter pinhole in a conical turret inlet (4 cm length, 3.4 cm ID; Fig. 1) at ∼ 7 slm into a stainless steel fluorescence cell, held at ∼ 2 hPa using a Roots blower (Leybold RUVAC WAU 1001) backed by a rotary pump (Leybold SOGEVAC SV200). An all solid-state laser system is used to excite OH via the A2 6 + (v 0 = 0) ← X2 53/2 (v 00 = 0) electronic transition at λ = 308 nm (HO2 is measured by conversion to OH using NO; see details below). The resultant fluorescence at 308 nm is detected by a micro-channel plate photomultiplier (MCP, Photek PMT325/Q/BI/G with 10 mm diameter photocathode) equipped with a 50 ns gating unit (Photek GM10https://doi.org/10.5194/amt-13-3119-2020 3121 Figure 1. Labelled SolidWorks model of the Leeds inlet pre-injector (IPI). The scavenger is injected into the centre of the perfluoroalkoxy (PFA) flow tube via four 0.25 mm ID needles. The thick yellow arrows indicate the direction of the sheath flow. 50) and a 2 GHz 20 dB gain amplifier (Photek PA200-10), and the signal is analysed by gated photon counting (Whalley et al., 2010). The background signal is normally obtained by scanning the laser wavelength off-resonance from the OH transition line, yielding the measurement commonly referred to as OHwave :   OH OH − Soffline = COH × S OH , (1) [OHwave ] = COH × Sonline where COH is the instrument calibration factor for OH and OH OH are the OH LIF signals at on- and offand Soffline Sonline resonance wavelengths, respectively. Similarly, the alternative measurement known as OHchem is defined as follows:   OH OH − Sonline, [OHchem ] = COH × Sonline scavenger , OH = COH × Sscavenger , (2) OH where Sonline, scavenger is the OH signal measured at an onresonance wavelength but in the presence of a scavenger. HO2 is detected via its conversion to OH following the addition of NO (BOC, 99.95 %, and Messer, 99.95 %). Although not reported here, RO2 radicals are measured using the ROx LIF method (Fuchs et al., 2008; Whalley et al., 2013), in which their reactions with NO and CO (BOC, 5 % in N2 , and Messer, 10 % in N2 ) result in conversion initially to OH (using NO; RO2 → HO2 → OH) and subsequently back to HO2 (using CO; OH → HO2 ) that is then detected as described above (via addition of NO inside the FAGE cell; HO2 → OH). The NO and CO are delivered using mass flow controllers (MFC, MKS Instruments 1179A series), which, unless otherwise stated, were also used to control all other gas flows described in this work. The Leeds FAGE instrument features two fluorescence cells, where laser light (∼ 10–20 mW at 308 nm, supplied at a pulse repetition frequency of 5 kHz via an optical fibre) enters each cell in Atmos. Meas. Tech., 13, 3119–3146, 2020 3122 R. Woodward-Massey et al.: Implementation of a chemical background method series. For normal operation in the field, the first cell (HOx ) measures OH and HO2 (low NO flow, 5 sccm; RO2 interference minimised) sequentially, while the second cell (ROx ) measures HO∗2 (high NO flow, 50 sccm; RO2 interference maximised) and then total RO2 (Fuchs et al., 2008; Whalley et al., 2013). Calibration of the FAGE instrument is achieved by supplying known radical concentrations via a turbulent flow tube (known in Leeds as the “wand”) held at ∼ 45◦ to the instrument inlet, where OH and HO2 are formed in a 1 : 1 ratio (Fuchs et al., 2011) by the photolysis of water vapour at 184.9 nm using a Hg(Ar) pen-ray lamp (LOT LSP035) in an excess flow (40 slm) of humidified ultra-high-purity air (BOC, BTCA 178, and Messer, 20.5 % O2 in N2 ). Chemical actinometry is performed via the photolysis of N2 O (BOC, medical-grade 98 %) to measure the product of lamp flux and photolysis exposure time to enable calculation of radical concentrations (Edwards et al., 2003; Faloona et al., 2004). The calibration of OH using the water vapour photolysis method has been validated by comparison with other methods, for example the kinetic decay of hydrocarbons (Winiberg et al., 2015). 2.2 Inlet pre-injector (IPI) design The Leeds inlet pre-injector (Fig. 1) is similar in concept to the design of Mao et al. (2012) and consists of a 4.0 cm length, 1.9 cm ID perfluoroalkoxy (PFA) cylinder embedded inside an aluminium housing, which seals to the FAGE cell via an O-ring base. The scavenger is injected into the centre of the PFA flow tube via four 0.25 mm ID needles, 4.0 cm above the FAGE inlet. The low bore capillary tubing increases the pressure inside the needles, which facilitates mixing of the scavenger into the ambient air stream. In this work propane (BOC, research-grade 99.95 %, and Messer, 99.995 %) was used as the main OH chemical scavenger, with similar results (see Sect. 3.1.2) obtained for C3 F6 (Sigma-Aldrich, 99 %). Based on previous investigations of OH interferences (Stevens et al., 1994; Dubey et al., 1996; Faloona et al., 2001; Ren et al., 2004; Mao et al., 2012; Griffith et al., 2013; Rickly and Stevens, 2018), C3 F6 was used initially as it reacts quickly and selectively with OH (k298 = 2.2 × 10−12 cm3 molec.−1 s−1 Sander et al., 2011) and does not contain any hydrogen atoms, which could serve as a source of laser-generated OH via abstraction by O(1 D) atoms (Stevens et al., 1994; Dubey et al., 1996). However, C3 F6 must be diluted in an inert gas before it can be flowed through MFCs, and its availability in the UK became limited in 2015. Following Novelli et al. (2014a), we therefore used propane for most laboratory experiments and all ambient measurements, despite the fact that it reacts more slowly with OH (k298 = 1.1×10−12 cm3 molec.−1 s−1 Sander et al., 2011). As shown in Fig. 2, the scavenger (0–50 sccm) is diluted in a flow of N2 (500 sccm, BOC, 99.998 %) prior to injection, Atmos. Meas. Tech., 13, 3119–3146, 2020 Figure 2. Diagram of the gas flows involved in IPI scavenger injection (not to scale). The two mass flow controllers (MFCs) are housed in the roof box, where the scavenger MFC (0–50 sccm) and injection valve (in a weatherproof housing on top of the roof box) are controlled using the main FAGE PC situated in the container laboratory. which is controlled using a solenoid valve (Metron Semiconductors). Any dead volume after the valve is purged continuously by the N2 dilution flow, using a narrow-bore injector inserted through the tee after the valve, with the injector tip placed as close to the valve orifice as possible. This enables fast flushing of the system to optimise the response time before and after scavenger injection. Incorporation of the purge system resulted in pre- and post-injection stabilisation times on the order of seconds (data not shown), minimising data loss. The valve state and scavenger flow over the course of the data acquisition cycle are controlled using a custom program nested within the FAGE software. To reduce radical wall losses, excess ambient air is drawn through the IPI to generate a sheath flow, via four ports spaced evenly around the flow tube housing as shown in Fig. 1. This minimises the FAGE sampling of air from near the walls of the cylinder, housing, and turret. The total flow rate through the IPI is 32 slm, of which 7 slm is sampled by the FAGE cell and the remainder of the flow is maintained by a vacuum pump (Agilent Technologies IDP-3 Dry Scroll Pump) and measured volumetrically using a rotameter (Brooks 2520, 4–50 L min−1 ). During interference testing experiments using the IPI system (Sect. 3.2), ozone and water vapour concentrations were measured using a commercial UV absorption instrument (Thermo Environmental Instruments Inc. 49C) and a chilled mirror dew point hygrometer (General Eastern 1311DR sensor and 4 × 4 Optica), respectively. https://doi.org/10.5194/amt-13-3119-2020 R. Woodward-Massey et al.: Implementation of a chemical background method 3123 Table 1. Overall meteorological and chemical conditions encountered during each field campaign, including example VOCs. Values are given as the median of all points that coincide with IPI sampling periods, except for J (O1 D) and O3 , which are reported as diurnally averaged maxima. Dates (IPI sampling) Location ICOZAa AIRPRO winterb 3–8 and 12–16 Jul 2015 52◦ 570 0200 N, 1◦ 070 1900 E 2–7 Dec 2016 23 May–25 Jun 2017 39◦ 580 2800 N, 116◦ 220 1600 E 16 1.5 5.8 16. 6.1 0.4 0.9 3.5 26 1.6 0.4 19 42 0.19 2.2 100 0.26 0.02 0.03 4.4 15 22. 33 1120 6.2 0.07 1.4 38 90 0.81 17 460 3.8 0.38 0.46 25 AIRPRO summer Meteorological Temperature (◦ C) H2 O (%) Wind speed (ms−1 ) J (O1 D) (10−6 s−1 ) Chemical O3 (ppbv) NO (ppbv) NO2 (ppbv) CO (ppbv) Propane (ppbv) Isoprene (ppbv) Benzene (ppbv) k’OH (s−1 ) a Integrated Chemistry of OZone in the Atmosphere. b An integrated study of AIR pollution PROcesses in Beijing (Shi et al., 2019). 2.3 Field measurement sites Ambient measurements of OHwave and OHchem were made using the Leeds IPI–FAGE instrument during three separate intensive field campaigns in different locations and seasons. This allowed for the investigation of potential OH interferences under markedly contrasting conditions. For all three field campaigns, measurements of OH, HO2 , and partially speciated RO2 were made using the Leeds FAGE instrument (4 m above ground level), operated in the sequential detection modes described in Sect. 2.1 (Whalley et al., 2013). To0 , was also measured, using the laser tal OH reactivity, kOH flash photolysis–LIF instrument described in detail by Stone et al. (2016). A range of supporting chemical, aerosol, and meteorological parameters were measured, with instruments situated either in buildings or shipping containers at each of the two sites. Gas-phase chemical observations included water vapour, NOx , NOy , O3 , CO, SO2 , HONO, HCHO (Cryer, 2016), ClNO2 , VOCs, and OVOCs. Photolysis rates (J ) for a variety of species, including O3 (→ O(1 D)), NO2 , HCHO, HONO, and ClNO2 , were measured using a 2π spectral radiometer (2π actinic receiver optic, Meteorologie Consult GmbH, coupled to an Ocean Optics QE Pro spectrometer), and J (O1 D) was also measured using a 2π filter radiometer (Meteorologie Consult GmbH) (Bohn et al., 2008). The meteorological and chemical conditions, including some example VOCs, encountered during each campaign are summarised in Table 1 and discussed in further detail below. https://doi.org/10.5194/amt-13-3119-2020 The first deployment of the Leeds IPI was during the ICOZA (Integrated Chemistry of OZone in the Atmosphere) project, which took place in July 2015, at the Weybourne Atmospheric Observatory (WAO), Weybourne, located on the northern Norfolk Coast, UK (52◦ 570 0200 N, 1◦ 070 1900 E, 15 m a.s.l.). The WAO is a Global Atmospheric Watch (GAW) regional station, and the site is impacted by a range of contrasting air masses, from clean Arctic air to processed emissions from the UK (e.g. London, which is located ∼ 180 km SSW of the observatory) and northern Europe. The aim of this field campaign was to improve understanding of ozone chemistry through integrated measurements of P (O3 ), i.e. the chemical or in situ ozone production rate (OPR) (Cazorla and Brune, 2010; Cazorla et al., 2012), with comparisons to a range of other observational and model approaches. Two continuous IPI sampling periods were conducted in the middle of the campaign, separated by a few days (3–8 and 12–16 July), with a total of 9 d where OHchem measurements are available around midday (fully continuous measurements were not always possible due to multiple instrument problems, e.g. cell blockages and laser power drops). For other times, only measurements of OHwave are available. During the IPI sampling periods, power cuts on the nights of 3–4 and 6–7 July resulted in extended data loss. In general, the ICOZA campaign was characterised (Table 1) by moderate temperatures (16 ◦ C median), high humidity (RH ∼ 80 %), and strong wind speeds (∼ 6 m s−1 ), as might be expected at a temperate, coastal location in the Atmos. Meas. Tech., 13, 3119–3146, 2020 3124 R. Woodward-Massey et al.: Implementation of a chemical background method summertime. The predominant wind sector, based on wind direction measurements at the site, was westerly (∼ 30 %), followed by southwesterly (∼ 20 %) and southerly (∼ 15 %). Back-trajectory analysis showed that during IPI sampling periods, the site was predominantly under the influence of Atlantic air (Cryer, 2016). These air masses had spent a considerable amount of time (∼ 1 d) over the UK, often encountering emissions from urban areas, which underwent photochemical ageing during their transport to the WAO site. Overall, the levels of pollution observed at the site were moderate, and the lowest of the three field campaigns discussed in this work (Table 1), and levels of isoprene were low. However, ozone mixing ratios were relatively high, with a dielaverage maximum of ∼ 40 ppbv, driven in part by strong UV and near-UV radiation. The Leeds IPI was deployed during another two campaigns at the Institute of Atmospheric Physics (IAP, 39◦ 580 36.0600 N, 116◦ 220 53.6900 E), an urban site in Beijing, China, during winter (November–December) 2016 and summer (May–June) 2017, as part of the AIRPRO (an integrated study of AIR pollution PROcesses in Beijing) project. AIRPRO is part of the wider APHH (Air Pollution and Human Health in a Chinese megacity) project (Shi et al., 2019), a joint UK–China programme. The aims of AIRPRO included the assessment of how pollutants are transformed and removed through transport, chemical, and photolytic processes, with a particular emphasis on the identification of the dominant oxidative degradation pathways (i.e. the relative importance of reactions with OH, NO3 , and O3 ). The AIRPRO project allowed for the assessment of OH measurement interferences under the highly polluted conditions of the megacity Beijing, situated on the heavily industrialised North China Plain. In winter, the site is impacted by urban and regional anthropogenic emissions, in particular those from the combustion of fossil fuels for residential heating. During summer, the site is subject to additional biogenic influences, and strong photochemical activity results in high rates of ozone production. In winter, OHwave and OHchem were measured simultaneously for 6 d of the campaign. In summer, almost 1 month of near-continuous IPI data are available, with 1 d of interruptions due to IPI testing (see Sect. 3.1.2). For both AIRPRO field intensives, the predominant wind sectors were westerly and southerly–southeasterly, which generally result in higher pollutant concentrations (Chen et al., 2015). Indeed, the two campaigns were subject to high pollutant concentrations, as illustrated by the elevated lev0 , many of which els of NO2 , CO, propane, benzene, and kOH were over an order of magnitude higher than ICOZA (Table 1). In addition, the biogenic influence during summer is clear from the relatively high isoprene concentrations observed, ∼ 0.4 ppbv on average but reaching up to 7.9 ppbv, a level considerably higher than those observed in some forested environments. Despite similar J (O1 D) values between ICOZA and AIRPRO summer, the higher VOC loadAtmos. Meas. Tech., 13, 3119–3146, 2020 ings during the latter resulted in increased production of ozone (90 ppbv diurnally averaged maximum). In contrast, AIRPRO winter was characterised by small ozone mixing ratios (15 ppbv diurnal maximum), as a consequence of high NO levels (median 22 ppbv) and weak UV radiation. In summer, NO levels were high in the morning (∼ 14 ppbv at 06:00 China standard time, CST) but surprisingly low in the afternoon, with diel-average median levels of ∼ 0.5 ppbv (15:00– 18:00 CST). For all ambient observation periods, the IPI data acquisition cycle consisted of 5 min of online wavelength and 30 s of offline wavelength (spectral background) integration, where the online period was split into 2 min of OH measurements and 2 min of propane addition to the IPI flow tube (chemical background), followed by 1 min of HO2 measurements (by the addition of NO to the FAGE cell). In terms of instrumental operation, the only difference between ICOZA and the AIRPRO campaigns was the use of different propane flows in the IPI. The propane concentration in the IPI flow 0 ∼ 3000 s−1 , τ tube was ∼ 110 ppmv (kOH OH ∼ 0.3 ms) during ICOZA and AIRPRO winter, but after internal removal experiments revealed that the propane level could be increased further (see Sect. 3.1.3), a 10-fold higher concentration (∼ 1100 ppmv), resulting in a concomitant reduction in the OH lifetime, was used for the AIRPRO summer campaign. On 1 d with high ozone (up to ∼ 80 ppbv) and moderate isoprene (∼ 0.5–1 ppbv) levels, the propane mixing ratio was reduced to ∼ 110 ppmv, but this had no observable effect on the background signals obtained for the summer data. All ambient OHwave data presented here have been corrected for the known interference from O3 in the presence of H2 O vapour (see Sect. 3.2.1). 3 Results 3.1 3.1.1 IPI characterisation Sensitivity The presence of additional surfaces in the IPI system may result in radical wall losses and therefore reduce the overall FAGE instrument sensitivity. To test for potential OH losses in the IPI flow tube, OH radicals were generated using a 184.9 nm Hg lamp placed ∼ 19 cm from the instrument inlet, so that ambient air with elevated radical concentrations (∼ 2– 7 × 107 molec. cm−3 ) was sampled, alternating between IPI and non-IPI sampling (Fig. 3), where for the latter the entire IPI assembly was removed. The dominant source of OH was the photolysis of ambient water vapour at 184.9 nm. In these experiments, the Hg lamp was placed sufficiently far away from each inlet within a large tent enclosure on the container roof, such that it could be assumed that OH concentrations were uniform in the region the inlet sampled from. Otherwise, the difference in inlet height between IPI and non-IPI https://doi.org/10.5194/amt-13-3119-2020 R. Woodward-Massey et al.: Implementation of a chemical background method 3125 Figure 3. Testing of OH losses in the IPI system. Each panel shows repeat measurements of OH signals (±2σ ) over the course of 1 d, where high OH concentrations were generated using a 184.9 nm Hg lamp placed near the instrument inlet. Blue and red markers denote individual measurements (one measurement “loop”, i.e. one wavelength online–offline cycle) performed with (“IPIon”) and without (“IPIoff”) the IPI system, respectively. Solid lines correspond to the average signals for each day, with 2σ standard deviations (SD) shown by the dashed lines. sampling may have resulted in different OH concentrations being sampled, e.g. due to differences in O3 absorption at 184.9 nm (O3 has a high cross section at this wavelength), which would affect the light flux at the point of sampling and hence the concentration of OH generated. Since ambient variability (e.g. in NOx levels) also affects the atmospheric radical concentrations, the IPI–non-IPI cycle was repeated several dozen times on 3 different days within the tent enclosure to ensure sufficient averaging of the results. Any differences in wind speed or direction during the different days are not important because of the tent enclosure. Based on the averages for each set of repeat measurements in Fig. 3, these experiments yield a mean ±2σ IPIoff/IPIon ratio of https://doi.org/10.5194/amt-13-3119-2020 1.043 ± 0.023, i.e. a < 5 % sensitivity reduction due to the presence of the IPI. While HO2 loss was not tested, the relative sensitivity is assumed to be closer to unity since it is less reactive than OH. In either case, the correction is smaller than the total instrumental uncertainty (∼ 26 % at 2σ ), and as such no corrections were applied to OH or HO2 calibration factors for the final workup of ambient data collected during IPI sampling periods. In other words, we assume negligible transmission losses within the IPI, and the OH calibration factor we applied to ambient data was the same for (1) OHwave without IPI sampling, (2) OHwave during IPI sampling, and (3) OHchem during IPI sampling. However, it Atmos. Meas. Tech., 13, 3119–3146, 2020 3126 R. Woodward-Massey et al.: Implementation of a chemical background method should be noted that in the field, calibrations are normally carried out without the IPI system present. The lack of OH loss in the IPI system is further supported by another test conducted in the field during the summer 2017 AIRPRO campaign, where on 1 d of the campaign sequential measurements of OHwave were taken with and without the IPI assembly present. While this was not a formal intercomparison, the summer 2017 campaign provided ideal conditions to assess IPI losses, considering the very high radical concentrations observed (OH was frequently > 1 × 107 molec. cm−3 ) in Beijing and thus a good signal-tonoise ratio. The results of this experiment are shown for OH in Fig. 4. It can be seen that if no correction for a reduction in sensitivity is applied, adjacent IPIoff and IPIon periods of data are qualitatively in agreement, with no discontinuities in the temporal profile, implying that the IPI sensitivity loss is close to zero under field operating conditions. Similar results were obtained for HO2 (data not shown). 3.1.2 External OH removal The external OH removal efficiency in the IPI system is controlled by the injection height, the choice of scavenger (i.e. the rate coefficient of the reaction of scavenger with OH), the scavenger and N2 dilution gas flows delivered to the injectors, and the sheath flow. A key requirement here is efficient mixing of the scavenger into the ambient air stream, which is difficult considering the fast flow rate and hence short residence time of air in the IPI flow tube. Additionally, it is important to consider that some reaction of the scavenger may occur inside the fluorescence chamber (internal OH removal, Sect. 3.1.3). This would give rise to a positive bias in ambient OH concentration measurements made using the OHchem method, as internal OH removal could result in loss of interfering OH and therefore an apparent reduction in the true background signal. External OH removal experiments were performed by supplying known concentrations of OH and HO2 to the instrument using the calibration wand described in Sect. 2.1. However, in contrast to normal calibration procedures where the wand is held at 45◦ to the pinhole (to overfill the pinhole and minimise sampling of pockets of air which may have been in contact with the metal pinhole surface), IPI characterisation experiments were performed with the wand positioned parallel to the direction of flow within the IPI (i.e. 90◦ relative to the plane of the pinhole), with a distance of ∼ 3 cm between the wand exit and the PFA flow tube. The high flow through the calibration wand (40 slm) ensured that an excess of calibration gas was delivered to the IPI system (sample flow ∼ 32 slm). The external OH removal efficiency (REOH external ) may be OH calculated from the proportion of OH remaining (Rexternal ) after injection of the scavenger, obtained from the ratio of the OH OH signals in the presence (Sscavenger ) and absence (S OH ) of Atmos. Meas. Tech., 13, 3119–3146, 2020 the scavenger: OH OH Rexternal (%) = 100 × Sscavenger /S OH , (3) OH REOH external (%) = 100 − Rexternal . (4) Initial tests included variation of the N2 dilution flow; however, the OH removal efficiency was generally low (data not shown), likely due to poor mixing of the scavenger into the sampled air when the flow rate from the injector is small. As a result, the N2 dilution was set to the maximum flow of the MFC used (0.5 slm) for all subsequent experiments. Any further dilution of the ambient air stream would result in a loss of sensitivity towards the detection of radicals; however, at 0.5 slm the dilution flow is virtually negligible compared to the total flow rate in the IPI system (32 slm). In other preliminary experiments, the scavenger was injected closer to the FAGE inlet (1.0 and 2.5 cm), but this also resulted in poor external OH removal owing to the shorter residence time between scavenger injection and FAGE sampling. The scavenging efficiency was determined for both propane and C3 F6 , with good agreement between the two scavengers. Figure 5 shows the remaining OH signal as a 0 =k function of the OH reactivity (kOH OH+scavenger [scavenger]) calculated in the flow tube, which normalises the scavenger concentrations according to their different reaction rates with OH. The observed removal efficiency is in broad agreement with the theoretical scavenging efficiency, based on the residence time in the flow tube (∼ 20 ms, assuming plug flow) and assuming perfect mixing, suggesting that in the Leeds IPI system the scavenger is well mixed into the gas sampled by the FAGE cell. An optimum removal of virtually 100 % (OH remaining ±2σ = (0.030 ± 0.091) %) 0 ∼ 3000 s−1 , equivalent to ∼ 110 ppmv was observed at kOH 15 (2.7×10 molec. cm−3 ) propane. This scavenger concentration was used for measurements of OHchem during the summer 2015 ICOZA project and winter 2016 AIRPRO project. For the summer 2017 AIRPRO project, a 10-fold higher scavenger concentration was used (∼ 1100 ppmv propane), after internal removal experiments revealed no loss of internal OH at this higher concentration, as discussed in detail in the next section. 3.1.3 Internal OH removal Internal removal of OH was quantified by Mao et al. (2012) after forming OH inside the PSU ground-FAGE cell using a Hg lamp and comparing the OH signal with and without the presence of the scavenger (C3 F6 ), which was added externally in the IPI system. It was found that most of the internal removal occurred in the instrument inlet, rather than in the OH detection axis, with a total loss of ∼ 20 %. Internal removal was not tested in the laboratory by Novelli et al. (2014a) for the Max Planck Institute for Chemistry (MPIC) FAGE instrument, but instead they limited the scavenger (propene and propane) concentration such that the exhttps://doi.org/10.5194/amt-13-3119-2020 R. Woodward-Massey et al.: Implementation of a chemical background method 3127 Figure 4. Time series of OHwave concentrations in Beijing on 17 June 2017, a period of high OH levels in the summer 2017 AIRPRO (an integrated study of AIR pollution PROcesses in Beijing) campaign. Blue and red markers (±1σ ) denote observations made with and without the IPI system, respectively. ternal OH removal efficiency was < 95 %, to minimise possible reaction of the scavenger with OH inside the fluorescence cell. However, during ambient, night-time tests (constant atmospheric OH concentration assumed), no change in the OH background signal was observed after increasing the scavenger concentration by a factor of 7, providing evidence for a lack of internal removal (Novelli et al., 2014a). In the present study, a novel approach was devised to quantify internal removal of OH in the Leeds IPI–FAGE instrument. First, under otherwise identical experimental conditions to those for external OH removal tests, sufficient CO (75 sccm, 95 ppmv) was added to the calibration wand to verify that the OH formed (alongside HO2 ) from the photolysis of water vapour was almost quantitatively converted to HO2 ((98.0 ± 0.4) %, data not shown). Secondly, in addition to the calibration wand CO flow, a high flow of NO (50 sccm) was injected through 0.125 in. stainless steel tubing inside the FAGE cell, with the injector tip positioned centrally just below the turret pinhole, to reconvert the HO2 back to OH for LIF detection; these experimental conditions ensured a fairly high HO2 -to-OH conversion efficiency of approximately 30 %. In this manner, OH was only generated inside the FAGE cell, and not in the IPI flow tube, such that any change in the fluorescence signal could be attributed to internal reaction of OH with propane, rather than reaction in the flow tube. The procedure for determination of internal OH removal bears some resemblance to that used for ambi- https://doi.org/10.5194/amt-13-3119-2020 ent detection of RO2 using the ROx LIF technique (Fuchs et al., 2008; Whalley et al., 2013), i.e. the external conversion of all radical species to HO2 before internal conversion to OH within the fluorescence cell. The internal OH removal efficiency (REOH internal ) was quantified in an analogous manner to the external scavenging efficiency, using the total fluorescence signal in the presence HOx (Sscavenger ) and absence (S HOx ) of the scavenger: OH HOx Rinternal (%) = 100 × Sscavenger /S HOx , (5) OH REOH internal (%) = 100 − Rinternal . (6) Figure 6 shows a time series of the LIF signal during two example internal removal experiments. Here, the LIF signal represents the sum of signals from OH and HO2 , since they are produced in a 1 : 1 ratio (Fuchs et al., 2011) in the calibration wand. For both of the propane mixing ratios used, which were shown to result in near complete external OH removal in Sect. 3.1.2, there was no obvious decrease in the LIF signal, indicating no significant internal removal of OH. The average ±2σ internal OH removal observed for repeat experiments was (0.0±4.0) % (Table 2) at a propane mixing ratio of ∼ 110 ppmv (ICOZA and AIRPRO winter conditions). For repeat experiments at the higher propane mixing ratio used during the AIRPRO summer field campaign (∼ 1100 ppmv), the internal removal was still very small and almost insignificant ((2.9 ± 6.6) %, Table 2). The observed internal OH removal may be compared to that which might be expected theAtmos. Meas. Tech., 13, 3119–3146, 2020 3128 R. Woodward-Massey et al.: Implementation of a chemical background method the assumption of constant gas velocity is invalid (i.e. the real residence time is closer to ∼ 1 ms) and mixing between HO2 and NO from the injector is not instantaneous. However, it should be noted that this calculation also neglects the fact that the density is higher in the jet and the perturbation to normal flow caused by moving the NO injector close to the pinhole. In the internal removal experiment, OH is not formed instantly at the pinhole but is built up linearly by HO2 /NO conversion along the line from the pinhole to the laser axis. Therefore, the experimental internal removal may not be directly compared with the theoretical estimate. In such a sequential reaction system, the OH scavenging is about half as efficient as that for the case where OH is formed as an instant point source at the pinhole. Thus, the experimental value should be doubled to (5.8 ± 13) %, which is in reasonable agreement with the theoretical value. From this, we cannot rule out a small internal OH removal on the order of 10 % at the higher propane level used for the AIRPRO summer campaign. However, no such corrections were applied to the ambient data featured in this work. 3.2 3.2.1 Figure 5. Proportion of the OH signal remaining (i.e. external OH removal efficiency) after addition of increasing concentrations of propane and perfluoropropene (C3 F6 ) scavengers to the IPI flow 0 ) to account for tube, converted to equivalent OH reactivities (kOH the different rate constants for the reaction of each scavenger with OH: (a) linear y scale, (b) log y scale. Error bars denote the 2σ SD of repeat experiments. The blue curve corresponds to the theoretical scavenging efficiency assuming perfect mixing, using the estimated residence time, τ ∼ 20 ms. The propane OH reactivity used for the ICOZA (Integrated Chemistry of OZone in the Atmosphere) and AIRPRO winter campaigns is given but that used for AIRPRO summer is off-scale. oretically. In the ambient pressure flow tube, a propane mix0 = 30 000 s−1 , but this ing ratio of 1100 ppmv equates to kOH is a factor of 760/1.5 lower in the detection cell (i.e. the ratio of ambient to cell pressure), 59 s−1 (assuming constant gas density and no change in the OH + propane rate coefficient). Under normal operation, NO injection occurs 10.5 cm below the pinhole and 7.5 cm away from the laser axis (i.e. total of 18 cm between the pinhole and detection volume), with a residence time of 0.9 ms between NO injection and OH detection (Creasey et al., 1997b; Whalley et al., 2013). The gas likely slows down between pinhole sampling and NO injection but, assuming a constant gas velocity, the residence time between the pinhole and the laser axis is estimated at ∼ 2 ms. Based on this, an internal OH removal efficiency of ∼ 12 % is calculated, which is higher than observed, likely because Atmos. Meas. Tech., 13, 3119–3146, 2020 Interference testing experiments O3 + H2 O vapour In LIF–FAGE instruments, there is a known interference due to laser-generated OH from ozone photolysis in the presence of water vapour (Fuchs et al., 2016; Griffith et al., 2016; Tan et al., 2017). This interference was quantified by Whalley et al. (2018) and characterised in further detail in the present work. In these experiments, ozone was generated from the 184.9 nm photolysis of oxygen in a 12–20 slm flow of zero air using a Hg(Ar) pen-ray lamp (LOT LSP035). Another 12–20 slm of zero air was humidified using a water (HPLCgrade) bubbler. The two zero air flows were combined and delivered to the calibration wand, from which the IPI sampled in a manner analogous to the experiments conducted to investigate external and internal OH removal discussed above. Ozone mixing ratios in the range 0–2.5 ppmv were generated by varying the Hg lamp current (0–21 mA), while water vapour volume mixing ratios in the range 0.1 %–1.0 % were produced by varying the flow through the bubbler, or by bypassing it completely, and the total flow (32 slm) was compensated for by changing the dry zero air flow. Laser power (LP) at 308 nm was varied in the range 3–17 mW by varying the ratio of acetone : water in a cuvette placed before the fibre launcher that is used to send laser light to the detection cells. Figure 7 shows the results of O3 + H2 O vapour interference tests. It can be seen that the interference signal (OHint = OHwave − OHchem ) is linear in both ozone (Fig. 7a) and water vapour (Fig. 7b) mixing ratios. The quadratic dependence of the interference signal on laser power (Fig. 7c) in terms of raw count rates indicates that the interference originates from a two-photon process, as expected. However, since OH https://doi.org/10.5194/amt-13-3119-2020 R. Woodward-Massey et al.: Implementation of a chemical background method 3129 Table 2. Internal removal of OH (%, ±2σ ) as a function of propane mixing ratio in the IPI flow tube, determined as shown in Fig. 6 (see text for details). Propane (ppmv) Experiment no. 110 (used for ICOZA and AIRPRO winter) 1 2 3 Weighted average ±2σ 1 1 2 3 Weighted average ±2σ 550 1100 (used for AIRPRO summer) Internal removal (%) −0.1 ± 4.8 0.3 ± 7.7 −0.9 ± 16 0.0 ± 4.0 1.0 ± 9.6 1.9 ± 12 4.2 ± 11 2.5 ± 11 2.9 ± 6.6 Figure 6. Time series of the LIF signal during internal OH removal experiments. The raw 1 s data are given by the grey line. NO was continuously added to the FAGE cell during these experiments (to form OH internally), and points where propane was added to the IPI flow tube are indicated by the orange-shaded panels, with the corresponding signal averages (±1σ ) shown as markers (see text for details). The first experiment (a) corresponds to the propane mixing ratio used for ICOZA, while the second (b) corresponds to that used for AIRPRO. The results of the internal OH removal experiments are summarised in Table 2. data are normalised to laser power, the equivalent OH concentrations are linear with respect to laser power. Thus, overall, OHint is linear in ozone, water vapour, and laser power. Normalisation of the slope in Fig. 7a yields the following relation: [OHint ] = (565 ± 42) molec. cm−3 ppbv−1 %−1 mW−1 × [O3 ] × [H2 O] × LP, (7) [O3 ] = 50 ppbv and [H2 O] = 1 %, and a typical instrument laser power of 15 mW, the interference signal is equivalent to an OH concentration of 3.9 × 105 molec. cm−3 . This signal is slightly smaller than the instrumental limit of detection (LOD) of ∼ 7 × 105 molec. cm−3 at a signal-to-noise ratio (SNR) of 2, but nonetheless it was used to correct the ambient OHwave data presented in Sect. 3.3, using co-located measurements of ozone and water vapour. where [O3 ], [H2 O], and LP are in units of ppbv, %, and mW, respectively. Under typical atmospheric conditions of https://doi.org/10.5194/amt-13-3119-2020 Atmos. Meas. Tech., 13, 3119–3146, 2020 3130 R. Woodward-Massey et al.: Implementation of a chemical background method Table 3. Summary of interference tests with O3 and isoprene (ISO) in the presence of propane (PROP), based on the data in Fig. 8. n/a – not applicable. Test A B C D OHint (molec. cm−3 ) O3 H2 O ISO PROP (ppmv) (%) (ppmv) (ppmv) Obs. Correction due to O3 + H2 O vapour interferencea OP3 levelsb AIRPRO 2017 levelsc 1.64 1.86 1.83 1.85 0.73 0.07 0.98 0.07 0 16 16 16 110 110 110 1100 1.0 × 107 1.9 × 107 1.6 × 107 1.4 × 107 0 1.8 × 107 8.0 × 105 1.3 × 107 n/a 21 1 15 n/a 430 19 310 a Corrected using Eq. (7). b Oxidant and Particle photochemical processes field campaign in Borneo, 2008: average O = 10 ppbv; ISO = 3.5 ppbv. 3 c Diurnally averaged maximum O = 90 ppbv; overall maximum ISO = 7.9 ppbv. 3 3.2.2 Isoprene ozonolysis To test for interferences from isoprene (ISO) ozonolysis products, isoprene (∼ 16 ppmv), and ozone (∼ 1.8 ppmv) were mixed in the calibration wand and the scavenger (propane, PROP) was injected into the IPI flow tube. The propane concentrations were set to those used for ambient OHchem measurements, such that the tests were representative of normal atmospheric sampling (i.e. to test whether an interference signal would remain in ambient data). However, to generate sufficient OH signal for quantitative analysis, ozone and isoprene were introduced at concentrations that far exceeded their typical ambient levels (Table 3). Unlike previous tests of interferences from alkene ozonolysis (Novelli et al., 2014b), low [O3 ] : [ISO] ratios were used to suppress the signal contribution from the atmospheric (real) OH generated by ozonolysis (i.e. isoprene acted as an additional OH scavenger). To allow sufficient time for steady-state conditions to develop, the IPI did not sample from the calibration wand directly, but instead a 30 cm flow tube (polycarbonate, ID ∼ 19 mm) was used to extend the IPI (which sampled wand gas at the normal IPI flow rate of ∼ 32 slm, residence time for O3 + isoprene reaction ∼ 0.15 s). Time series of the interference testing experiments conducted using the IPI are shown in Fig. 8. In Fig. 8a, no isoprene is added, but due to ozone photolysis in the presence of high [H2 O] (0.73 %) an interference signal (OHint ) is observed (i.e. signal in the presence of propane is higher than the offline signal). The magnitude of this signal (OHint ∼ 1.0 × 107 molec. cm−3 ) yields a scale factor of 510 ± 270 molec. cm−3 ppbv−1 %−1 mW−1 when linearly extrapolated down from the measured [O3 ], [H2 O], and LP, in agreement with the 565 ± 42 molec. cm−3 ppbv−1 %−1 mW−1 in Eq. (7). In Fig. 8b, ozone and isoprene react under dry conditions, and an interference signal is observed again. The low H2 O (0.07 %) suppressed the O3 + H2 O interference, such that this cannot explain the magnitude of OHint (∼ 1.9 × 107 molec. cm−3 , Table 3), suggesting that OH was formed Atmos. Meas. Tech., 13, 3119–3146, 2020 internally from a reaction other than O1 D + H2 O. Under high-humidity (H2 O ∼ 1 %) conditions (Fig. 8c), OHint (∼ 1.6 × 107 molec. cm−3 ) was similar, but in this case the signal can be explained almost entirely by the O3 +H2 O interference. Under dry conditions with a 10-fold higher concentration of propane (as used for the AIRPRO summer campaign, Fig. 8c), the interference signal from Fig. 8b was reduced but remained elevated relative to the offline signal (OHint ∼ 1.4×107 molec. cm−3 ), where again the contribution from O3 + H2 O cannot explain the discrepancy. The decrease in OHint between Fig. 8b and d may be attributed to the suppression of steady-state OH generated from ozonolysis, but the remaining signal implies that OH was also formed internally in both cases. For the dry, low-propane experiment (Fig. 8b), the magnitude of the OH signal is much higher than that calculated from a steady-state model (∼ 1.4×106 molec. cm−3 ). The suppression of the interference signal attributable to O3 /isoprene only (i.e. O3 + H2 O corrected) by the addition of water vapour (H2 O ∼ 1 %; see Fig. 8c) suggests that the internal OH may have been formed from SCIs. The simplest C1 and C2 SCIs are known to react quickly with the water vapour dimer (k ∼ 4–7 × 10−12 cm3 molec.−1 s−1 at 298 K for CH2 OO; Chao et al., 2015; Lewis et al., 2015) and monomer (k ∼ 1–2 × 10−14 cm3 molec.−1 s−1 for antiCH3 CHOO; Taatjes et al., 2013; Sheps et al., 2014; Lin et al., 2016), respectively. Reaction with the water vapour monomer was also shown to be relatively fast (k ∼ 1.2 × 10−15 cm3 molec.−1 s−1 , kloss ∼ 300 s−1 at ∼ 1 % H2 O) for the ensemble of SCIs, including the C1 SCI, generated from isoprene ozonolysis (Newland et al., 2015). However, regardless of whether the signal observed at high propane is due to internally formed OH, which may have originated from SCIs, the equivalent OH concentrations are negligible when extrapolated back to ambient chemical conditions (Table 3). Assuming a linear dependence of the interference signal on both ozone and isoprene, the interference (after O3 + H2 O correction) is equivalent to < 102 molec. cm−3 at the ozone (10 ppbv) and isoprene (3.5 ppbv) levels measured in a low-NOx , biogenic environ- https://doi.org/10.5194/amt-13-3119-2020 R. Woodward-Massey et al.: Implementation of a chemical background method 3131 Figure 7. OH interference (±1σ ) from O3 + H2 O as a function of (a) O3 , (b) H2 O, and (c) laser power. The interference signal is linear in O3 and H2 O mixing ratios and quadratic in laser power, confirming the two-photon nature of the process. Normalising the slope in panel (a) to O3 = 1 ppbv, H2 O = 1 %, and laser power = 1 mW yields an OH interference equivalent to a concentration of 565 ± 42 molec. cm−3 . ment during the Oxidants and Particle photochemical processes (OP3) campaign in Borneo, 2008 (Hewitt et al., 2010). Similarly, we have modelled the SCI decomposition in our FAGE cell. We assumed an ambient atmosphere containing 100 ppbv O3 and 10 ppbv trans-2-butene and took the reactions, rate coefficients, and yields from Novelli et al. (2014a) and MCMv3.3.1. The rate of CI to SCI was 1 × 106 s−1 with an SCI yield of 0.18. The unimolecular decomposition of SCI to OH was 3 s−1 and the wall loss rate of SCI was 22 s−1 . From this we calculate an equivalent ambient pressure OH concentration of ∼ 4 × 103 molec. cm−3 from the decomposition of SCIs at our FAGE cell residence time of 2 ms. The insignificance of the interference signal for atmospherically relevant O3 /alkene concentrations is consistent with the results of previous interference experiments, for which equivalent OH concentrations of ∼ 3–4×104 (Novelli et al., 2014b; https://doi.org/10.5194/amt-13-3119-2020 Fuchs et al., 2016) and ∼ 4 × 105 molec. cm−3 (Rickly and Stevens, 2018) can be derived. 3.2.3 NO3 radicals Fuchs et al. (2016) found that, despite the absence of a hydrogen atom, NO3 radicals were responsible for a small OH interference signal in the Forschungszentrum Jülich (FZJ) LIF–FAGE instrument, equivalent to an OH concentration of 1.1×105 molec. cm−3 per 10 pptv NO3 . The OH interference scaled linearly with observed NO3 mixing ratios but showed no dependence on inlet length, cell pressure, laser power, or humidity, and the background signal did not change significantly in the presence of CO scavenger, suggesting the OH was indeed being formed internally. It was postulated that the interference was a result of a heterogeneous process involvAtmos. Meas. Tech., 13, 3119–3146, 2020 3132 R. Woodward-Massey et al.: Implementation of a chemical background method Figure 8. Isoprene (ISO) ozonolysis interference tests: (a) O3 /H2 O only, (b) O3 and ISO under dry conditions, (c) O3 and isoprene with H2 O added, and (d) O3 and isoprene under dry conditions but with a higher concentration of propane (PROP) to remove any steady-stategenerated OH. Shaded areas are periods of propane addition, and the light blue lines correspond to the calculated signals from O3 +H2 O only (for experiments with isoprene present). The interference signals (“OH no. 2” – “offline”) were used to derive equivalent OH concentrations (OHint ), which are on the order of ∼ 1–2 × 107 molec. cm−3 . These experiments are summarised in Table 3. See text for further details. Atmos. Meas. Tech., 13, 3119–3146, 2020 https://doi.org/10.5194/amt-13-3119-2020 R. Woodward-Massey et al.: Implementation of a chemical background method 3133 Figure 9. Equivalent OH concentrations (±1σ ) measured during NO3 radical interference tests. NO3 concentrations were calculated using a box model, and OH interference signals were corrected for the interference from O3 + H2 O. The OH limit of detection (LOD, 6.3 × 105 molec. cm−3 , SNR = 2) is denoted by the dashed red line. ing NO3 and H2 O adsorbed on instrument walls. Interference signals were also observed in the detection of HO2 and RO2 radicals, equivalent to 1.0 × 107 and 1.7 × 107 molec. cm−3 , respectively, per 10 pptv NO3 . To test for an NO3 interference in OH measurements made by the Leeds FAGE instrument, NO3 was generated from the reaction of ozone and NO2 : O3 + NO2 → NO3 , (R1) NO3 + NO2 → N2 O5 , (R2) N2 O5 → NO3 + NO2 . (R3) In these experiments, ozone was generated by flowing zero air (15 slm) past a Hg lamp (LOT LSP035). A constant 0.5 slm flow of NO2 (BOC, 2 ppmv) was diluted in 25 slm zero air and mixed with the zero air–ozone flow and an additional zero air dilution flow of 10 slm, in order to yield a final mixing ratio of 20 ppbv. Gas was delivered to the IPI system using the calibration wand, with a total residence time of 3.7 s for the O3 + NO2 reaction. Ozone mixing ratios in the range 0–2.8 ppmv (after dilution) were generated by varying the current supplied to the Hg lamp. NO3 radical mixing ratios in the range 0–90 pptv were calhttps://doi.org/10.5194/amt-13-3119-2020 culated based on a box model with rate constants taken from the Master Chemical Mechanism (MCM; http://mcm. leeds.ac.uk/MCM, last access: 15 December 2017) version 3.3.1 (kR1 = 3.52 × 10−17 cm3 molec.−1 s−1 , kR2 = 1.24 × 10−12 cm3 molec.−1 s−1 , and kR3 = 0.045 s−1 ). These experiments were performed under dry conditions (H2 O ∼ 0.07 %), such that only a small correction was applied for the O3 /H2 O interference. The results of the NO3 radical interference tests are shown in Fig. 9. It can be seen that the equivalent OH signals were all < 8 × 105 molec. cm−3 and almost always below the instrument LOD of 6.3 × 105 molec. cm−3 (SNR = 2). Unlike the dependence found by Fuchs et al. (2016), the interference signal does not increase linearly with NO3 . However, based on the point at the highest NO3 mixing ratio of ∼ 90 pptv, the interference is equivalent to an insignificant ∼ 6×104 molec. cm−3 at 10 pptv NO3 , or approximately half of that observed by Fuchs et al. (2016). These experiments suggest that an interference from NO3 radicals is not significant for the detection of OH using the Leeds ground-based FAGE instrument. Atmos. Meas. Tech., 13, 3119–3146, 2020 3134 R. Woodward-Massey et al.: Implementation of a chemical background method Figure 10. Overall intercomparison of OHwave and OHchem observations from the ICOZA campaign. Grey markers represent raw data (4 min), with 1 h averages (±2 standard error, SE) in red. The thick red line is the orthogonal distance regression (ODR) fit to the hourly data, with its 95 % confidence interval (CI) bands given by the thin red lines; fit errors are given at the 2σ level. For comparison, 1 : 1 agreement is denoted by the dashed blue line. OHwave data were corrected for the known interference from O3 + H2 O. 3.3 3.3.1 Ambient observations of OHwave and OHchem ICOZA 2015 Figure 10 shows the overall intercomparison of OHwave (with O3 + H2 O interference as given in Eq. 7 subtracted) and OHchem measurements made during the ICOZA 2015 campaign. It is evident that the raw data (averaged for 4 min periods) are quite noisy, but averaging to 1 h improves the precision and reveals a tight correlation, with the majority of points scattered around the line of 1 : 1 agreement. An orthogonal distance regression (ODR) fit (Boggs et al., 1987), which accounts for errors in both the y and x directions, to the hourly data yields a slope of 1.160 ± 0.058 (2σ ) and a negative intercept on the order of the instrumental precision. In a similar manner, an unweighted least-squares linear fit (not shown) gives a slope of 1.060 ± 0.065, an intercept of (0.5 ± 1.5) ×105 molec. cm−3 , and a correlation coefficient (R 2 ) of 0.992. Regardless of the fit method, these results show that, on average, the two OH measurements agree within the instrumental uncertainty of ∼ 26 % at 2σ . Median hourly diurnal profiles of OHwave , OHchem , and J (O1 D), averaged over both IPI sampling periods, are shown in Fig. 11. The two OH measurements exhibit virtually identical profiles, with peak values of ∼ 3 × 106 molec. cm−3 slightly before solar noon, and relatively high concentrations (∼ 1–2 × 106 molec. cm−3 ) persisting into the early evening despite the concomitant falloff in J (O1 D). Night-time levels Atmos. Meas. Tech., 13, 3119–3146, 2020 were generally below 5×105 molec. cm−3 . The variability in OH concentrations, shown only for OHchem for clarity, was high during both daytime and night-time periods. In Fig. 10 it can be seen that some points lie substantially above the 1 : 1 line, especially for the 4 min averaged raw data. It is possible that, despite the good overall agreement between the median diurnal profiles of OHwave and OHchem in Fig. 11, OHint may have exhibited its own distinct diurnal profile, independent of atmospheric OH concentrations, for example if the interference signal was generated from a particular chemical species. However, the median diurnal profile of individual OHint measurements (= OHwave – OHchem ) in Fig. 11 exhibits no obvious structure, with values scattered around zero and a mean ±2σ value of (0.3 ± 3.3) ×105 molec. cm−3 , which is well below the LOD for individual OH measurements. Similarly, the average (OHwave − OHchem ) / OHwave ratio (i.e. the contribution of interferences to the total OHwave signal) was zero within error (mean ±2 standard error, SE, = 0.03 ± 0.12). Furthermore, OHint does not exhibit any dependence when binned against various parameters (Fig. S1 in the Supplement), including those previously implicated in LIF–FAGE measurement interferences, such as J (O1 D) (Feiner et al., 2016); temperature (Mao et al., 2012; Novelli et al., 2017); OH reactivity (Mao et al., 2012); and O3 (Feiner et al., 2016; Novelli et al., 2017), isoprene (Feiner et al., 2016), and NO (Feiner et al., 2016) mixing ratios. https://doi.org/10.5194/amt-13-3119-2020 R. Woodward-Massey et al.: Implementation of a chemical background method 3135 Figure 11. Hourly median diurnal profiles of OHwave , OHchem , and J (O1 D) (right axis) from the ICOZA campaign. Also shown (red line and markers) is the hourly median diurnal profile of OHint (= OHwave − OHchem ), calculated from individual 4 min data points; the single red marker corresponds to the average (±2σ ) of this trace. The variability (interquartile range, IQR) in OHchem measurements is denoted by the dashed grey lines and is not shown for others for clarity. OHwave data were corrected for the known interference from O3 + H2 O. 3.3.2 AIRPRO winter 2016 The overall agreement between the two measurements is presented in the correlation plot in Fig. 12. As with ICOZA (Fig. 10), a tight correlation is revealed after averaging the data to 1 h, and all points are distributed evenly around the line of 1 : 1 agreement. ODR fitting yields an overall slope of 1.051 ± 0.039 and a negative intercept of a similar magnitude to the instrumental precision. An unweighted leastsquares linear fit (not shown) gives a slope of 0.997 ± 0.038, an intercept of (5.1 ± 7.3) ×104 molec. cm−3 , and an R 2 of 0.97. The two measurements exhibit the same profile on a diurnal basis (Fig. 13), with a diel maximum of ∼ 3 × 106 molec. cm−3 occurring in the late morning due to the build-up of HONO overnight. At night, OHchem concentrations were close to the LOD (< ∼ 2 × 105 molec. cm−3 ), while OHwave measurements were frequently negative, possibly as a result of over-subtraction of the O3 /H2 O interference as this is subject to high uncertainty (Fig. 7). The diurnal profile of OHint is scattered around zero with a mean ±2σ difference of (−0.9 ± 2.7) ×105 molec. cm−3 , and the mean ±2 SE contribution of interferences to the total signal was −0.02 ± 0.07. https://doi.org/10.5194/amt-13-3119-2020 3.3.3 AIRPRO summer 2017 The intercomparison of OHwave and OHchem measurements for the AIRPRO summer campaign is shown in Fig. 14. Consistent with ICOZA and the AIRPRO winter results, the 1 h data are scattered around the 1 : 1 line, with an overall ODR slope of 1.103 ± 0.017. However, the intercept is more negative than for the other campaigns, which suggests that the O3 /H2 O interference may have been overestimated because it is during this campaign that the highest ozone mixing ratios (∼ 90 ppbv diurnally averaged maximum, Table 1) were encountered. Similarly, an unweighted least-squares linear fit to the data (not shown) yields a slope of 1.111±0.029, an intercept of (−3.8±1.7) ×105 molec. cm−3 , and an R 2 of 0.92 (data not shown). Again, the two measurements follow the same diurnal profile (Fig. 15), peaking in the afternoon at ∼ 1 × 107 molec. cm−3 with relatively high night-time levels of ∼ 1–2 × 106 molec. cm−3 . As with ICOZA and the AIRPRO winter campaign, the OHint diurnal profile does not exhibit any obvious structure, with values scattered around zero and a mean ±2σ difference of (−1.6 ± 4.1) ×105 molec. cm−3 . The mean ±2 SE (OHwave – OHchem ) / OHwave ratio was −0.09 ± 0.10. During AIRPRO, measured NO3 mixing ratios reached up to ∼ 100 pptv, such that the lack of significant night-time OH interference signals is consistent with the results of NO3 interference tests (Sect. 3.2.3). Atmos. Meas. Tech., 13, 3119–3146, 2020 3136 R. Woodward-Massey et al.: Implementation of a chemical background method Figure 12. Overall intercomparison of OHwave and OHchem observations from the winter 2016 AIRPRO campaign. Grey markers represent raw data (4 min), with 1 h averages (±2 SE) in red. The thick red line is the ODR fit to the hourly data, with its 95 % CI bands given by the thin red lines; fit errors are given at the 2σ level. For comparison, 1 : 1 agreement is denoted by the dashed blue line. OHwave data were corrected for the known interference from O3 + H2 O. Figure 13. Hourly median diurnal profiles of OHwave , OHchem , and J (O1 D) (right axis) from the winter 2016 AIRPRO campaign. Also shown (red line and markers) is the hourly median diurnal profile of OHint , calculated from individual 4 min data points; the single red marker corresponds to the average (±2σ ) of this trace. The variability (IQR) in OHchem measurements is denoted by the dashed grey lines and is not shown for others for clarity. OHwave data were corrected for the known interference from O3 + H2 O. Atmos. Meas. Tech., 13, 3119–3146, 2020 https://doi.org/10.5194/amt-13-3119-2020 R. Woodward-Massey et al.: Implementation of a chemical background method 3137 Figure 14. Overall intercomparison of OHwave and OHchem observations from the summer 2017 AIRPRO campaign. Grey markers represent raw data (4 min), with 1 h averages (±2 SE) in red. The thick red line is the ODR fit to the hourly data, with its 95 % CI bands given by the thin red lines; fit errors are given at the 2σ level. For comparison, 1 : 1 agreement is denoted by the dashed blue line. OHwave data were corrected for the known interference from O3 + H2 O. Figure 15. Hourly median diurnal profiles of OHwave , OHchem , and J (O1 D) (right axis) from the summer 2017 AIRPRO campaign. Also shown (red line and markers) is the hourly median diurnal profile of OHint , calculated from individual 4 min data points; the single red marker corresponds to the average (±2σ ) of this trace. The variability (IQR) in OHchem measurements is denoted by the dashed grey lines and is not shown for others for clarity. OHwave data were corrected for the known interference from O3 + H2 O. https://doi.org/10.5194/amt-13-3119-2020 Atmos. Meas. Tech., 13, 3119–3146, 2020 3138 R. Woodward-Massey et al.: Implementation of a chemical background method It can be seen from Fig. 14 that there is a small cluster of points that lie significantly away from both the 1 : 1 and ODR regression lines, which are characterised by high OHwave concentrations of > 1.5 × 107 molec. cm−3 . This finding was investigated further, with the results summarised in Fig. 16. Above an OHchem threshold of ∼ 1.0–1.5×107 molec. cm−3 , the OHint signal becomes significantly greater than zero and the instrument LOD, reaching ∼ 3–5 × 106 molec. cm−3 at OHchem levels of around ∼ 2 × 107 molec. cm−3 . However, these results should be treated with caution, since only a few points are available for which OHchem was present at such high concentrations. The same behaviour was not observed for either the AIRPRO winter or ICOZA campaigns, since OHchem levels did not surpass 1 × 107 molec. cm−3 , but the analogous mean values at low OHchem concentrations are in agreement with the AIRPRO summer results. The above results suggest that in the Beijing summertime, the Leeds FAGE instrument is subject to an interference(s) at the highest OH levels, although its contribution of ∼ 15 %– 20 % (Fig. 16) is still below the instrumental accuracy of 26 % at 2σ . This finding is consistent with the suggestion of Fittschen et al. (2019) that ROOOH species, formed from RO2 + OH reactions, generate an OH interference in LIF– FAGE instruments, since high OH levels would generate high RO2 concentrations and favour this class of reaction. It is also possible that, for high ambient OH production rates, the scavenger cannot react with the sampled OH sufficiently quickly, leading to elevated but spurious OHchem background signals. Although a modelling study of the inlet chemistry would be required to fully assess this hypothesis, it is likely not the case considering that the propane concentration used during AIRPRO results in an OH lifetime of ∼ 0.03 ms in the IPI flow tube, in comparison to a residence time of ∼ 20 ms (i.e. ∼ 700 OH lifetimes). No clear dependences were found when OHint was binned against various parameters previously implicated in OH measurement interferences (Fig. S2), although it can be seen that OHint was marginally higher in the highest temperature, J (O1 D), and isoprene bins. 4 Discussion The results from the three field campaigns that feature in this work demonstrate that, in moderately to highly polluted conditions, the Leeds ground-based FAGE instrument does not suffer from substantial interferences in the measurement of OH using the conventional, wavelength modulation background technique, OHwave . This is illustrated best by the slopes of the overall measurement intercomparison plots (Figs. 10, 12, and 14), which ranged from 1.05 to 1.16. However, while the deviations of these slopes from 1 are small, they are still significant, suggesting the presence of unknown OH interferences. Nonetheless, such unknown interferences are well within the instrumental uncertainty of ∼ 26 % at 2σ . Atmos. Meas. Tech., 13, 3119–3146, 2020 With respect to previous studies during which OH has been measured by a LIF instrument equipped with a scavenger injector, the significance of interferences during the campaigns that feature in this work are amongst the lowest observed (Table 4). This can likely be attributed to two main factors: environment and instrumental. In terms of the former, none of the field campaigns described in the present study took place in forested environments, where the most significant interferences have been observed (Mao et al., 2012; Novelli et al., 2014a; Feiner et al., 2016). However, as mentioned previously, the AIRPRO summer campaign did share some characteristics, in that high BVOC and low NO mixing ratios were observed in the afternoon. Despite this, OHwave and OHchem were in good agreement. Although AIRPRO summer took place in a city, its results do provide confidence in previous measurements of OH using the same instrument, and support the hypothesis that there are unknown OH sources in the atmosphere. The insignificance of daytime interferences during the AIRPRO campaigns is consistent with results of another urban study, CalNex-LA (Research in California at the Nexus of Air Quality and Climate Change) (Griffith et al., 2016). The O3 /H2 O interference is much higher (up to ∼ 4 × 106 molec. cm−3 OH equivalent during CalNexLA) in the Indiana University (IU) LIF instrument (Dusanter et al., 2009), such that the daytime contributions of ∼ 33 % can be explained entirely by this known interference. However, measurements made at a nearby site during the same study (CalNex-SJV) showed daytime contributions of ∼ 20 % (Brune et al., 2016), although this may be related to instrumental differences, as discussed below. On average, interferences were not observed in the daytime during ICOZA, but they were observed in other coastal campaigns, namely DOMINO (Diel Oxidants Mechanisms In relation to Nitrogen Oxide), HOx (∼ 50 %) (Novelli et al., 2014a), and CYPHEX (CYprus PHotochemistry EXperiment, ∼ 45 %) (Mallik et al., 2018), as well as in rural regions, such as HOPE (Hohenpeißenberg Photochemistry Experiment, 20 %–40 %) (Novelli et al., 2014a). Studies of the North China Plain have revealed small interferences on the order of 0 %–10 % (Tan et al., 2017, 2018), with slightly higher but variable contributions of 0 %–20 % in the Pearl River Delta (Tan et al., 2019). The second major reason for the differences in contributions between the studies listed in Table 4 is likely instrumental effects. For the campaigns in which the highest OH interferences have been observed (Mao et al., 2012; Novelli et al., 2014b; Feiner et al., 2016), OH measurements were made using the Max Planck Institute (MPI) (Martinez et al., 2010) and PSU (Faloona et al., 2004) LIF instruments. These instruments feature laser multi-pass detection cells, which give rise to larger detection volumes and increased UV fluence, although this may not be relevant considering that the interference signals did not display any laser power dependence for these instruments. The Leeds instrument also differs in https://doi.org/10.5194/amt-13-3119-2020 R. Woodward-Massey et al.: Implementation of a chemical background method 3139 Figure 16. The relationship between OHint and OHchem using binned data. The error bars for AIRPRO summer (a, blue markers) denote 1 SD and are not shown for AIRPRO winter and ICOZA for clarity. The dashed black line corresponds to a sigmoid fit and is used to guide the eye only. The contribution of interferences to the total OHwave signal (= OHint / OHwave ×100 %) for the AIRPRO summer campaign is shown in (b), with the number of points in each bin shown in (c). All OHint data used here have been corrected for the known interference from O3 + H2 O. terms of cell geometry, where the HOx cell is composed of a short (5 cm) turreted inlet on top of a large fluorescence cell (additional ∼ 8 cm to laser axis, ∼ 13 cm total length, and a cell diameter of 25 cm). In contrast, the MPI and PSU instruments feature flow-tube-like inlets (14–17 cm from the pinhole to laser axis) mounted on smaller fluorescence cells, facilitating the interaction of sampled gas with the cell walls, which may promote the generation of internal OH. For the measurements listed in Table 4, the Peking University (PKU) instrument (Tan et al., 2017) is most similar to the Leeds FAGE (i.e. single-pass detection, ∼ 10 cm total length from sampling inlet to laser axis), for which similar daytime interferences on the order of ∼ 0 %–20 % were observed. For the ICOZA campaign, nothing could be inferred about the origin of the OH interference signal when one was observed, as it did not exhibit any characteristic diurnal profile (Fig. 11), and showed no obvious dependences on a variety of meteorological and chemical parameters. This finding is in contrast to previous studies in which diel profiles (Mao et al., 2012; Feiner et al., 2016) and dependences (Mao et https://doi.org/10.5194/amt-13-3119-2020 al., 2012; Feiner et al., 2016; Novelli et al., 2017) of the interference have been observed. The occurrence of large (i.e. > 1×106 molec. cm−3 ) background OH signals (OHint = OHwave − OHchem ) after instrumental problems (e.g. power cuts, data not shown) implies that the differences may have been instrumental rather than as a result of a species present in ambient air, although the data at these times did pass all quality control filters and therefore could not be rejected. Nonetheless, any differences are still a concern, regardless of their cause; the IPI system thus serves as an additional check on measurement accuracy and operational stability, and is perhaps most useful for fieldwork sites where power supplies are unreliable, for example in more remote areas. It is possible that, even though the background OH had a flat diurnal profile in each field campaign, the species responsible for any interference observed were different between daytime and night-time periods. Thus, analysis of the daytime and night-time data separately, as a function of the same parameters, might reveal more information. Considering the recent identification of NO3 radicals as an internal Atmos. Meas. Tech., 13, 3119–3146, 2020 3140 R. Woodward-Massey et al.: Implementation of a chemical background method Table 4. Average contributions of FAGE background signals to the total OH measured (= (OHwave – OHchem ) / OHwave ) during ambient air studies where a chemical modulation technique was employed. Study Year PROPHET BEARPEX CABINEX SHARP CalNex-LA CalNex-SJV DOMINO HOx 1998 2009 2009 2009 2010 2010 2010 HUMPPA-COPEC Location Environment type Contribution (%) Daytime Night-time Reference(s) Forest, isoprene-dominated Forest, MBO-dominateda Forest, isoprene-dominated Urban Urban, downwind of LA Urban Coastal, close to petrochemical industry Boreal forest, terpene-dominated Not tested 40–60 Not tested 30 33b 20 50 ∼0 50 50–100 50 Not reported 80 100 Faloona et al. (2001) Mao et al. (2012) Griffith et al. (2013) Ren et al. (2013) Griffith et al. (2016) Brune et al. (2016) Novelli et al. (2014) 2010 N Michigan NE California N Michigan Houston, Texas Pasadena, California Bakersfield, California El Arenosillo, near Huelva, SW Spain Hyytiälä, SW Finland 60–80 100 HOPE SOAS Wangdu 2012 2013 2014 Hohenpeissenberg, S Germany Near Brent, Alabama North China Plain Rural Forest, isoprene-dominated Rural, urban influenced 20–40 80 10 100 > 70 Not reported CYPHEX 2014 NW Cyprus 45 100 PRIDE-PRD2014 BEST-ONE ICOZA AIRPRO Winter 2014 2016 2015 2016 Pearl River Delta North China Plain N Norfolk Coast, UK Beijing, China Coastal, influenced by processed European emissions Suburban, 60 km SW of Guangzhou Suburban, 60 km NE of Beijing Coastal, London outflow Urban Hens et al. (2014); Novelli et al. (2014, 2017) Novelli et al. (2014, 2017) Feiner et al. (2016) Fuchs et al. (2017); Tan et al. (2017) Mallik et al. (2018) <8 ∼0 ∼0 ∼0 AIRPRO Summer 2017 Beijing, China Urban ∼0 0–20 ∼0 ∼0 Night-time OH almost always < LOD ∼0 Tan et al. (2019) Tan et al. (2018) This work This work This work a MBO = 2-methyl-3-buten-2-ol, a biogenic volatile organic compound. b Consistent with known O + H O interference. 3 2 OH source in LIF instruments (Fuchs et al., 2016), and that OH concentrations have often been under-predicted at night (Faloona et al., 2001; Mao et al., 2012; Ren et al., 2013; Hens et al., 2014; Lu et al., 2014; Tan et al., 2017), this is perhaps the most interesting period for further study. However, for the data presented in this work, robust quantitative night-time analyses are not possible due to OH measurements being below or close to the instrument LOD. In this work, there are several key findings that stand out. First, OHwave and OHchem were in good agreement even at the very low NO concentrations of < 100 pptv during ICOZA and the moderate afternoon levels (∼ 500 pptv on average but often < 100 pptv; Shi et al., 2019) during the AIRPRO summer campaign. While the role of isoprene could not be assessed for ICOZA, due to the limited range of concentrations observed (< 0.2 ppbv), it reached high levels during AIRPRO summer (up to 7.9 ppbv, larger than seen in some forested regions) but did not seem to perturb the agreement between the two measurements. In addition, very high levels of aromatic VOCs were observed during both AIRPRO winter and summer, where the agreement between OHwave and OHchem suggests that the intermediates of aromatic oxidation, such as exotic bicyclic species (Birdsall et al., 2010) and highly oxygenated molecules (HOMs) (Wang et al., 2017; Molteni et al., 2018; Tsiligiannis et al., 2019), do not give rise to OH interferences, which is postulated to be the case for intermediates (SCIs) in the ozone-oxidation of alkenes (Novelli et al., 2014b, 2017; Rickly and Stevens, 2018). However, the large alkene and ozone concentrations observed during AIRPRO summer should favour the formation of these SCIs, but sig- Atmos. Meas. Tech., 13, 3119–3146, 2020 nificant interferences were not observed, consistent with laboratory investigations of the isoprene interference and casting doubt on the SCI hypothesis. Although, the AIRPRO SCI concentrations also depend on the magnitude of the SCI loss rates, which could be high if elevated levels of SO2 (Welz et al., 2012; Sheps et al., 2014) or organic acids (Welz et al., 2014) were present. Considering the success of the first three field deployments of the IPI system and given that it does not reduce the instrument sensitivity towards OH, it is suggested that the system is adopted for permanent use in ambient studies, although conventional sampling should still be performed from time-totime to check for potential artefacts caused by the IPI system itself. Another advantage of the IPI system is that it reduces the amount of solar light entering the pinhole, which reduces the size and variability of daytime background signals and therefore improves signal-to-noise and hence detection limits. It is recommended that the IPI propane concentration is kept the same as the summer AIRPRO campaign, as it is possible that the slightly poorer agreement between OHwave and OHchem during ICOZA was because of the lower propane flow used (i.e. the flow was not sufficient to ensure that OH generated from all steady-state sources was removed), although this cannot be verified. Future field campaigns using the IPI will allow for the assessment of interferences in the Leeds FAGE instrument for a range of different environments. From these, the contribution of interferences for previous studies in similar environments, where measurements were made prior to the discovery of significant interferences in the LIF measurement of OH reported https://doi.org/10.5194/amt-13-3119-2020 R. Woodward-Massey et al.: Implementation of a chemical background method by others, may be inferred. The measurement–model comparisons may then be reassessed in light of any new information regarding the accuracy of OH measurements. Regardless of the reasons for any differences between the two measures of OH (i.e. chemical interferences or instrumental problems such as during recovery periods after power cuts), the IPI system serves as an additional check on OH observations, increasing confidence in the validity of the data obtained. 5 Conclusions The addition of an IPI system to the Leeds ground-based FAGE instrument allowed for a comprehensive investigation of OH measurement interferences in both the laboratory and the field. Following its optimisation and thorough characterisation in terms of sensitivity and external and internal OH removal efficiency, laboratory experiments were conducted to assess potential interferences from (1) the photolysis of O3 in the presence of H2 O vapour, (2) the intermediates and products of isoprene ozonolysis, and (3) NO3 radicals. For O3 + H2 O, a small but potentially significant interference (at high O3 levels) was found, but interferences from isoprene ozonolysis products and NO3 radicals were shown to be insignificant under typical atmospheric conditions. Field campaigns conducted in the UK and China showed that, on average, the Leeds ground-based FAGE instrument does not suffer from significant interferences in the detection of OH. It was only under the very high OH levels of > 1.5 × 107 molec. cm−3 sometimes observed during the AIRPRO summer campaign that interferences were found consistently, although their contributions (∼ 15 %–20 %) were smaller than the instrumental accuracy of 26 % at 2σ . Large interference signals (> 1 × 106 molec. cm−3 ) were occasionally observed during the ICOZA campaign but always after instrumental problems such as power cuts, suggesting that the OHchem method serves as an additional tool for verifying instrument stability and validating measurements. The Leeds IPI system will find continued use in future fieldwork. Data availability. Data presented in this study are available from the authors upon request (l.k.whalley@leeds.ac.uk and d.e.heard@leeds.ac.uk). Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/amt-13-3119-2020-supplement. Author contributions. RWM, TI, LKW, and DEH designed the IPI system. RWM, EJS, JA, LMS, CY, and LKW performed laboratory experiments. RWM, EJS, DRC, CY, LKW, and DEH were responsible for field measurements. RWM, EJS, and LKW analysed the data. RWM wrote the manuscript with input from all co-authors. https://doi.org/10.5194/amt-13-3119-2020 3141 Competing interests. The authors declare that they have no conflict of interest. Acknowledgements. Robert Woodward-Massey, Danny R. Cryer, and Eloise J. Slater are grateful to NERC for funding PhD studentships. We would like to acknowledge Brian Bandy, Grant Forster, David Oram, and Claire Reeves (University of East Anglia); William Bloss, Leigh Crilley, and Louisa Kramer (University of Birmingham); and Rachel Dunmore, Jacqui Hamilton, James Hopkins, James Lee, Chris Reed, and Freya Squires (University of York) for the provision of some of the data used to generate the averages in Table 1. We would also like to thank other participants in the ICOZA and AIRPRO field campaigns. 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W4250065189.txt

Qeios · Definition, February 2, 2020 Ope n Pe e r Re v ie w on Qe ios Beta Adrenergic Receptor National Cancer Institute Source National Cancer Institute. Beta Adrenergic Receptor. NCI T hesaurus. Code C17062. Expressed by Beta Adrenergic Receptor Genes in effector tissues innervated by postganglionic sympathetic adrenergic fibers, Beta Adrenergic Receptors are one of two major classes of adrenergic receptors (alpha and beta) based on their reactions to norepinephrine and epinephrine, on cellular effects of receptor activation, and on relative affinities and reactions to synthetic blocking or stimulating agents. Beta-adrenergic receptors respond to blocking agents such as propranolol and to activating agents such as isoproterenol. Beta-1 type receptors mediate lipolysis and increase cardiac rate and force of contraction; beta-2 type receptor activation promotes bronchial and vascular smooth muscle relaxation. When coupled to adenylate cyclase through Gs protein, Beta Adrenergic Receptor stimulation results in increased intracellular cyclic AMP. (NCI) Qeios ID: 0JO81Z · https://doi.org/10.32388/0JO81Z 1/1 

W4376277518.txt

Field Efficacy of Various Insecticides and Trichogramma Chilonis (Ishii) Against Tomato Fruit Borer Helicoverpa Armigera (Hubner) (Lepidoptera: Notctuidae) Amani Mulk Khan (  Amanimulkkhan@yahoo.com ) The University of Agriculture Peshawar Faculty of Crop Protection Sciences Ahmad-Ur-Rahman Saljoqi Mehran Ullah Muhammad Amin Research Article Keywords: Posted Date: May 12th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2871672/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/19 Abstract The present study investigated the comparative efficacy of botanical extracts (Neem 2 and 3%), commercial insecticide (Radiant) and bio-agent (Trichogramma chilonis) against tomato fruit borer Helicoverpa armigera in tomato crop under field conditions, Agriculture Research Institute (ARI) Tarnab Peshawar, 2019. The experiment was laid out under randomized complete block design (RCBD) with factorial arrangement having five treatments (Radiant, Neem (2 and 3%) and Trichogramma chilonis) replicated three times. Pre spray data was recorded before 24hrs of treatment application while post spray data was recorded after different time intervals (24hrs, 48hrs, 72hrs, 5 days, 7days, 10 days and 15 days) of each treatment application. Data regarding infestation showed that highest percent reduction over control was recorded in Radiant treated plots followed by Neem 3% and Trichogramma treated plots. The efficacy of each treatment was recorded at peak after 7 days then gradually declined and was found minimum after 15 days interval. However, highest larval and eggs infestation were recorded in control plots throughout the experimental period. Statistical analysis of the data also revealed that botanical extracts (Neem 2 and 3%), commercial insecticide (Radiant) and bio agent (Trichogramma chilonis) significantly reduced percent fruit damage. Highest percent reduction in comparison with control was recorded in the plots treated with commercial insecticide Radiant. Data further revealed that each treatment application significantly increased total tomato yield and total marketable tomato yield. Highest tomato total marketable yield (9583 kg ha-1) with 80% increase over control was recorded in the Radian treated plots. It can be concluded that Radiant, Trichocards and Neem 3% comparatively have a high potential in reduction of fruit infestation which as a result gives higher yield with high percentage of marketable fruits and with highest cost benefit ratio. Introduction Tomato (Lycopersicon esculentum Mill) belongs to family Solanacea, is the most popular and widely cultivated vegetable in the world. It can be used in fresh or in processed form, while it is also a good source of vitamins A, B and C and aids in wound healing due to its antibiotic properties (Baloch 1994). According to the Food and Agriculture Organization corporate statistical data base, In 2017 production of tomato in the world was 182,301,395 metric tons. The largest producer country was China accounting 33% of total world production followed by India, USA and Turkey (FAOSTAT 2018). Statistics of 2015– 2016 shows that the total area under cultivation of tomato in Pakistan was 4542 ha produce total of 142462 tons tomato, while in Khyber Pakhtunkhwa the total production of tomato was 43931 tons from the area of 3708 ha. (MNFSR 2016-17). As compared to other countries the production of tomato is low in Pakistan due to several reasons while insect pests cause huge losses. Thus the quality and quantity of tomato is damaged at different level by different insect pests such as Aphids, jassids, whitefly and fruit worms (Hoffmann 2007). The important insect pest of tomato are fruit worm, Helicoverpa armigera (Hubner), and is the most serious agricultural insect pests worldwide (Atwal 1976). Page 2/19 In Pakistan, among other insect pests Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) is one of the important pests of many cultivated plants like tomato, chick pea, cotton, pigeon pea and are regularly the victims of H. armigera in various areas of the country. Tomato fruit borer feeds gregariously at the larval stages and due to this feeding habit causes huge loses in production of fruit. Adult of the borer usually can be seen from April to May and lays its eggs on the leaves. The immature larva feeds on the leaves, flowers and tomato fruits and finishes its developmental stages in the fruit. Larvae have almost 6 instars, all levels of larvae damage tomato fruit. In the process of maturation, larvae can comes out and move to another tomato fruit. The last instar of larvae falls to the ground from tomato fruit and pupates beneath the soil. Approximately 12 fruits of tomato can be damaged by one caterpillar, reduce market value, and ultimately cause a great deal of financial damage to farmers (Hussain and Bilal (2007). To deal with this potential and notorious pest, 85% of the world use insecticide to manage this pest (Shaheen 2008). However, incredible use of synthetic insecticide has led to the problem of pest resurgence, insecticidal resistance in insects, environmental pollution, health problems, decimation of useful fauna and the increased cost of pest controls as well (Hussain 1993). In order to address all these issues, it has become imperative to explore the substitutes of these toxic insecticides to keep the H. armigera population below injury level and have no harmful effect on bio control agents (Hassan 1992). Biological control is becoming a promising pest control method with many advantages over insecticides such as host-specific, non-toxic benefit to mammals and organisms, less insect-resistant, readily biodegradable and cheaper as well (Wink 1993). Similarly, there has been great interest in the development of environmental friendly botanical formulations (Schmutterer 1990). These include neem (Azadirachta indica), Ageratum, Chrysanthemum and Karanj, which have been used to control Helicoverpa pest generations. It has become the most powerful source of botanical pesticides (Schmutterer 1990). Trichogramma is an ovarian parasite species most frequently used in a large-scale biological surveillance program against lepidopteran pests. Adult of T. chilonis (Hymenoptera: Trichogrammatidae) are small wasps that kills a host's eggs by letting one or more of its eggs into much larger Lepidopteran eggs. Different species of Trichogramma are maximally used insects in the world because of their ease of mass production and application against many pests (Ayvaz et al. 2008). People use different type of pesticides to combat these pests and save crops. However, the continued and widespread use of similar insecticide groups causes problems with pesticide residues and other environmental pollution. This has highlighted the need to develop new, safer and easily degradable pesticides that can be viable and effective for pest control. Spinosad is one of those new substances derived from the soil actinomycete fermentation soup Saccharopolyspora spinosa, which comprise of spinosyn A and spinosyn D. Spinosad have faster contact and ingestion activity in insects that helps in causing excitation of the nervous system, leading to paralysis and cessation of feeding (Amalendu et al. 2009). Page 3/19 The economic importance of agricultural products has forced farmers to use pesticides almost daily, sometimes as much as twice according to the recommended dose. The indispensable use of synthetic pesticides to control this pest has led to the formation of harmful pesticides and fruit residues (Armes et al. 1994). METHODS AND MATERIALS Location The study regarding field efficacy of botanical, commercial insecticide and Trichogramma chilonis against tomato fruit borer Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) was conducted in the field of Entomology section, Agriculture Research institute (ARI) Tarnab Peshawar, from April to August 2019. Field Layout, Preparation and Transplantation The experiment was laid out under Randomized Complete Block Design (RCBD) with five treatments replicated three times. The whole field was divided into three equal parts considered as Blocks or Replicates. Then each block was further divided into five subplots 3x4 meter each by leaving 1 meter space/distance between these subplots to act as buffer zone, to avoid mixing or drift of the tested treatments. All standard agronomic practices required for vegetable growing before nursery transplantation also including recommended dose of NPK fertilizer application was applied at the time of field preparation. Before plantation five ridges were prepared in each subplot with row to row distance of 75 cm. When the nursery of tomato plants acquired the desired width, that is common pencil size and with 5–6 inch height, during March it was transplanted. Plant to plant distance was kept 30 cm throughout the replications. In order to avoid possible movement of T. chilonis, plots treated with T. chilonis were covered with green house net having height of 1.5 meter throughout the experiment. Treatments Selected treatments shown in the following tables were used against tomato fruit borer (H. armigera). Treatments were prepared on the basis of recommended dose. Treatments Common name Trade name Active ingredient Recommended dose 1 Spinetoram Radiant Spinetoram 0.8ml/L 2 Neem oil Neem oil 2% Azadirichta indica 20ml/L 3 Neem oil Neem oil 3% Azadirichta indica 30ml/L 4 Trichogramma Trichogramma chilonis ---------------- 5 cards/12m2 Page 4/19 1. Trichogramma chilonis (Ishii) egg cards Trichocards each containing 50 numbers of parasitized eggs of T. chilonis was acquired from the laboratory of entomology ARI, Tarnab, Peshawar. Trichocards were taken in the plastic jar and staple with randomized selected plants in each plot before the initiation of flowering stage. 2. Neem Oil Neem oil was purchased from the local market and was applied at 2% and 3% concentration respectively. 3. Radiant 120SC Radiant is the main product of Arysta life sciences, having spinetoram as active ingredient was purchased from the local market of Khyber Pakhtunkhwa, Peshawar, Pakistan. 4. Control Only water was sprayed on control plots and they was remained untreated as well. Application of the Treatments Trichocards each having 50 numbers of parasitized eggs at pupal stage of Trichogramma was installed in the field. Trichocards containing more emergence potential in less time was acquired from the laboratory. Five Trichocards, each containing 50 number of parasitized eggs was counted and was applied/stappled before initiation of flowering stage with the tomato leaves in per plot. Application of botanical and chemical insecticide were started as soon as the pest infestation appeared and reached to ETL (1 larvae/plant) till the end of the crop. When the first symptoms of the pests was seen at the beginning of flowering, botanical (neem oil) and insecticide (radiant) treatments were used, followed by a second and third application at an interval of 15 days. Spray of each treatment was applied during normal weather conditions particularly not during just afternoon and windstorm so that to avoid drift. Parameters The data was recorded on the basis of following parameters. 1. Larval population count The number of larvae per plant was observed separately, by using the forceps, soft camel hair brush and petri dishes or direct count method. Pre spray data was recorded 24hr before the spray application and then after spray application at the interval of 24 hours, 48 hours, 5th day, 7th day, 10th day and 15th day for each plant. Samples were taken in polyethylene bags to bring laboratory and examined by Page 5/19 stereomicroscope. All preserved specimens were identified at Plant Protection Department, with taxonomic keys. The infestation for each replicated plot was averaged and presented in the Table. 2. Pest population count The pest population including larval density and egg counts on each leaf of the plant was recorded through magnifying lens. Eggs infestation data was recorded before 24 hrs of spray application and then after spray application at interval of 24 hours, 48 hours, 5th day, 7th day, 10th day and 15th day. 3. Percent fruit damage per treatment 3.1 Percent fruit damage by weight The percent fruit damage by weight was calculated before and after spray application separately and then the damage fruits were added and converted by applying the following formula. Fruit damage by weight = weight of damaged fruit x 100/ total weight of fruit 3.2 Percent fruit damage by number The percent of damage fruit by its number was noted down distinctly and then converted all the damage fruit into percent by the following applied formula. Fruit damage by number = number of damage fruits x 100/ total number of fruits 4. Effect of each treatment on yield of tomato The effect of each treatment on the yield of tomato was determined by adding the total tomato fruit picked in replicated plots for each treatment as per the following formula. 5. Cost Benefits Ratio Analysis: Cost benefit ratio shows the utmost economic control measure for management of different pest in various crops. The cost benefit ratio for all treatments was calculated by the following formula. Cost benefit ratio = Total income/Total cost Statistical analysis Page 6/19 All the data was subjected to analysis of variance (ANOVA) under RCBD to evaluate the infestation of H. armigera larval and egg population. Means were separated using LSD tested at 0.05% level of significance using statistix 8.1 package (Steel and Torrie, (1980). RESULTS Field efficacy of botanicals, synthetic insecticide and bio control agent were tested against tomato fruit borer (Helicoverpa armigera) on tomato crop. Data were obtained for various parameters including cost benefit ratio and presented in Tables 1–9 with their ANOVA given in Appendix I-I0. Results recorded are described below. First Spray 1. Effect on eggs infestation Effect of botanical, commercial insecticides and Trichogramma on mean eggs infestation of fruit borer H. armigera after 1st spray at different time intervals was recorded. The mean data after 1st spray are presented in Table 1. Table 1 Effect of botanical, commercial insecticides and Trichogramma on eggs infestation of H. armigera after 1st spray at different time intervals Treatments 24 hrs 48 hrs 72 hrs 5 days 7 days 10 days 15 days Mean Radiant 3.97e 3.86fg 3.77ijkl 3.67mno 3.56r 3.58qr 3.61opqr 3.72c Neem 3% 3.96e 3.89f 3.79hijk 3.72lm 3.60qr 3.62opqr 3.64nopq 3.75b Trichogramma 3.90f 3.88f 3.82ghi 3.74kl 3.61pqr 3.63nopq 3.66nop 3.75b Neem 2% 3.86fg 3.79hijk 3.80hij 3.76jkl 3.64nopq 3.66nop 3.68mn 3.74b Control 3.84fgh 3.90f 3.98de 4.04d 4.10c 4.32b 4.57a 4.11a Mean 3.91a 3.86b 3.83c 3.79d 3.70e 3.76d 3.83c *Means in rows and columns followed by different letters are significantly different at P-value ≤ 0.05 LSD value (P ≤ 0.05) for treatment = 0.022 LSD value (P ≤ 0.05) for time = 0.026 LSD value (P ≤ 0.05) for treatment× time intervals = 0.057 Statistical analysis of the data revealed that both botanical, commercial insecticides and Trichogramma significantly (P ≤ 0.05 and F-value = 56.87) reduced the eggs population of H. armigera. Lowest eggs population (3.72 eggs/plant) was recorded in the plots treated with commercial insecticide Radiant, followed by eggs population (3.75 eggs/plant) was recorded in the plots treated with botanical insecticide Page 7/19 Neem (3%). The eggs population (3.75 eggs/plant) was noted in the plots treated with biological agent (Trichogramma). Highest eggs population (4.11 eggs/plant) was observed in control treatment. Different time intervals also significantly (P ≤ 0.05 and F-value = 468.97) reduced the eggs population of H. armigera. Lowest eggs population (3.70eggs/plant) was recorded after 7 days, followed by the eggs population (3.76 eggs/plant) was recorded after 10 days. Highest eggs population (3.91 eggs/plant) was recorded after 24 hours. Interaction effect of time interval and treatments were also found significant (P ≤ 0.05 and F-value = 65.46). Lowest eggs population (3.56 eggs/plant) was recorded after 7 days in the plots treated with commercial insecticide Radiant, followed by the eggs population (3.58 eggs/plant) was recorded after 10 days in the plots treated with Radiant. Highest eggs population (4.57 eggs/plant) was recorded after 15 days in control plots. 2. Effect on larval infestation The mean data regarding larval population density of H. armigera after application of first spray are presented in Table 2. Table 2 Effect of botanical, commercial insecticides and Trichogramma on larval population count of H. armigera after 1st application at different time intervals Treatments 24 hrs 48 hrs 72 hrs 5 days 7 days 10 days 15 days Mean Radiant 2.12e 2.01fg 1.92ijkl 1.82mno 1.71r 1.73qr 1.76opqr 1.87c Neem 3% 2.11e 2.04f 1.94hijk 1.87lm 1.75qr 1.77opqr 1.79nopq 1.90b Trichogramma 2.05f 2.03f 1.97ghi 1.89kl 1.76pqr 1.78nopq 1.81nop 1.90b Neem 2% 2.01fg 1.94hijk 1.95hij 1.91jkl 1.79nopq 1.81nop 1.83mn 1.89b Control 1.99fgh 2.05f 2.13de 2.19d 2.25c 2.47b 2.72a 2.26a Mean 2.06a 2.01b 1.98c 1.94d 1.85e 1.91d 1.98c *Means in rows and columns followed by different letters are significantly different at P-value ≤ 0.05 LSD value (P ≤ 0.05) for treatment = 0.022 LSD value (P ≤ 0.05) for time = 0.026 LSD value (P ≤ 0.05) for treatment× time intervals = 0.057 Statistical analysis of the data revealed that both botanical, commercial insecticides and Trichogramma significantly (P ≤ 0.05 and F-value = 56.87) reduced the larval population of H. armigera. Lowest larval population (1.87 larvae/plant) was recorded in the plots treated with commercial insecticide Radiant, Page 8/19 followed by larval population (1.90 larvae/plant) was recorded in the plots treated with botanical insecticide Neem (3%). The larval population (1.90 larvae/plant) was noted in the plots treated with bio control agent (Trichogramma). Highest larval population (2.26 larvae/plant) was observed in control treatment. Different time intervals also significantly (P ≤ 0.05 and F-value = 468.97) reduced the larval population of H. armigera. Lowest larval population (1.85 larvae/plant) was recorded after 7 days, followed by the larval population (1.91 larvae/plant) was recorded after 10 days. Highest larval population (2.06 larvae/plant) was recorded after 24 hours. Interaction effect of time interval and treatments were also found significant (P ≤ 0.05 and F-value = 65.46). Lowest larval population (1.71 larvae/plant) was recorded after 7 days in the plots treated with commercial insecticide Radiant, followed by the larval population (1.73 larvae/plant) was recorded after 10 days in the plots treated with Radiant. Highest larval population (2.72 larvae/plant) was recorded after 15 days in control plots. Second spray 1. Effect on eggs infestation Effect of botanical, commercial insecticides and Trichogramma on mean eggs infestation of fruit borer (H. armigera) after 2nd spray at different time intervals was recorded. The mean data after 2nd spray are presented in Table 3. Page 9/19 Table 3 Effect of botanical, commercial insecticides and Trichogramma on eggs infestation of H. armigera after 2nd spray at different time intervals Treatments 24 hrs 48 hrs 72 hrs 5 days 7 days 10 days 15 days Mean Radiant 3.58ij 3.50lm 3.37o 3.30pqr 3.16v 3.20uv 3.24stu 3.34e Neem 3% 3.62hi 3.54kl 3.37o 3.29pqr 3.20uv 3.23tu 3.26rst 3.36d Trichogramma 3.64gh 3.58jk 3.46mn 3.37o 3.24stu 3.28qrs 3.31pq 3.41c Neem 2% 3.67g 3.61hij 3.50lm 3.42n 3.30pqr 3.33op 3.36o 3.46b Control 4.66f 4.76e 4.84d 4.96c 5.06b 5.14a 5.18a 4.94a Mean 3.83a 3.80b 3.71c 3.67d 3.59f 3.64e 3.67d *Means in rows and columns followed by different letters are significantly different at p-value ≤ 0.05 LSD value (P ≤ 0.05) for treatment = 0.016 LSD value (P ≤ 0.05) for time = 0.019 LSD value (P ≤ 0.05) for treatment× time intervals = 0.042 Statistical analysis of the data revealed that both botanical, commercial insecticides and Trichogramma significantly (P ≤ 0.05 and F-value = 172.40) reduced the eggs population of H. armigera. Lowest eggs population (3.34 eggs/plant) was recorded in the plots treated with commercial insecticide Radiant, followed by eggs population (3.36 eggs/plant) was recorded in the plots treated with botanical insecticide Neem (3%). The eggs population (3.41 eggs/plant) was noted in the plots treated with biological agent (Trichogramma). Highest eggs population (4.94 eggs/plant) was observed in control treatment. Different time intervals also significantly (P ≤ 0.05 and F-value = 15290.7) reduced the eggs population of H. armigera. Lowest eggs population (3.59eggs/plant) was recorded after 7 days, followed by the eggs population (3.64 eggs/plant) was recorded after 10 days. Highest eggs population (3.83 eggs/plant) was recorded after 24 hours. Interaction effect of time interval and treatments were also found significant(P ≤ 0.05 and F-value = 108.65). Lowest eggs population (3.16 eggs/plant) was recorded after 7 days in the plots treated with commercial insecticide Radiant, followed by the eggs population (3.20 eggs/plant) was recorded after 10 days in the plots treated with Radiant. Highest eggs population (5.18 eggs/plant) was recorded after 15 days in control plots. 2. Effect on larval infestation Effect of botanical, commercial insecticides and Trichogramma on mean larval population count of fruit borer (H. armigera) after 2nd spray at different time intervals was recorded. The mean data after 2nd Page 10/19 spray are presented in Table 4. Table 4 Effect of botanical, commercial insecticides and Trichogramma on larval population count of H. armigera after 2nd spray at different time intervals Treatments 24 hrs 48 hrs 72 hrs 5 days 7 days 10 days 15 days Mean Radiant 1.73ij 1.65lm 1.52o 1.45pqr 1.31v 1.35uv 1.39stu 1.49e Neem 3% 1.77hi 1.69kl 1.52o 1.44pqr 1.35uv 1.38tu 1.41rst 1.50d Trichogramma 1.79gh 1.73jk 1.61mn 1.52o 1.39stu 1.43qrs 1.46pq 1.56c Neem 2% 1.82g 1.76hij 1.65lm 1.57n 1.45pqr 1.48op 1.51o 1.61b Control 2.81f 2.91e 2.99d 3.11c 3.21b 3.29a 3.33a 3.09a Mean 1.99a 1.95b 1.86c 1.82d 1.74f 1.79e 1.82d *Means in rows and columns followed by different letters are significantly different at p-value ≤ 0.05 LSD value (P ≤ 0.05) for treatment = 0.016 LSD value (P ≤ 0.05) for time = 0.019 LSD value (P ≤ 0.05) for treatment× time intervals = 0.042 Statistical analysis of the data revealed that both botanical, commercial insecticides and Trichogramma significantly (P ≤ 0.05 and F-value = 172.40) reduced the larval population of H. armigera. Lowest larval population (1.49 larvae/plant) was recorded in the plots treated with commercial insecticide Radiant, followed by larval population (1.50 larvae/plant) was recorded in the plots treated with botanical insecticide Neem (3%). The larval population (1.56 larvae/plant) was noted in the plots treated with biological agent (Trichogramma). Highest larval population (3.09 larvae/plant) was observed in control treatment. Different time intervals also significantly (P ≤ 0.05 and F-value = 15290.7) reduced the larval population of H. armigera. Lowest larval population (1.74larvae/plant) was recorded after 7 days, followed by the larval population (1.79 larvae/plant) was recorded after 10 days. Highest larval population (1.99 larvae/plant) was recorded after 24 hours. Interaction effect of time interval and treatments were also found significant (P ≤ 0.05 and F-value = 108.65). Lowest larval population (1.31 larvae/plant) was recorded after 7 days in the plots treated with commercial insecticide Radiant, followed by the larval population (1.35 larvae/plant) was recorded after 10 days in the plots treated with Radiant. Highest larval population (3.33 larvae/plant) was recorded after 15 days in control plots. Third spray Page 11/19 1. Effect on eggs infestation Effect of botanical, commercial insecticides and Trichogramma on mean eggs infestation of fruit borer (H. armigera) after 3rd spray at different time intervals was recorded. The mean data after 3rd spray are presented in Table 5. Table 5 Effect of botanical, commercial insecticides and Trichogramma on eggs infestation of H. armigera after 3rd spray at different time intervals Treatments 24 hrs 48 hrs 72 hrs 5 days 7 days 10 days 15 days Mean Radiant 3.22hi 3.00mno 2.76t 2.57u 2.41v 2.44v 2.47v 2.70e Neem 3% 3.27h 3.11jk 3.02lmn 2.90pq 2.76t 2.78st 2.80rst 2.95d Trichogramma 3.30gh 3.16ij 3.10jkl 3.00mno 2.86qrs 2.88pqr 2.92opq 3.03c Neem 2% 3.36g 3.27h 3.16ij 3.07klm 2.96nop 3.00mno 3.02lmn 3.12b Control 5.27f 5.42e 5.56d 5.69c 5.74c 5.84b 5.96a 5.64a Mean 3.68a 3.59b 3.52c 3.45d 3.35f 3.39e 3.43d *Means in rows and columns followed by different letters are significantly different at p-value ≤ 0.05 LSD value (P ≤ 0.05) for treatment = 0.033 LSD value (P ≤ 0.05) for time = 0.039 LSD value (P ≤ 0.05) for treatment× time intervals = 0.088 Statistical analysis of the data revealed that both botanical, commercial insecticides and Trichogramma significantly (P ≤ 0.05 and F-value = 73.24) reduced the eggs population of H. armigera. Lowest eggs population (2.70 eggs/plant) was recorded in the plots treated with commercial insecticide Radiant, followed by eggs population (2.95 eggs/plant) was recorded in the plots treated with botani1cal insecticide Neem (3%). The eggs population (3.03 eggs/plant) was noted in the plots treated with biological agent (Trichogramma). Highest eggs population (5.64 eggs/plant) was observed in control treatment. Different time intervals also significantly (P ≤ 0.05 and F-value = 10652.0) reduced the eggs population of H. armigera. Lowest eggs population (3.35eggs/plant) was recorded after 7 days, followed by the eggs population (3.39 eggs/plant) was recorded after 10 days. Highest eggs population (3.68 eggs/plant) was recorded after 24 hours. Interaction effect of time interval and treatments were also found significant (P ≤ 0.05 and F-value = 44.01). Lowest eggs population (2.41 eggs/plant) was recorded after 7 days in the plots treated with Page 12/19 commercial insecticide Radiant, followed by the eggs population (2.44 eggs/plant) was recorded after 10 days in the plots treated with Radiant. Highest eggs population (5.96 eggs/plant) was recorded after 15 days in control plots. 2. Effect on larval infestation Effect of botanical, commercial insecticides and Trichogramma on mean larval population count of fruit borer (H. armigera) after 3rd spray at different time intervals was recorded. The mean data after 3rd spray are presented in Table 6. Table 6 Effect of botanical, commercial insecticides and Trichogramma on larval population count of H. armigera after 3rd spray at different time intervals Treatments 24 hrs 48 hrs 72 hrs 5 days 7 days 10 days 15 days Mean Radiant 1.37hi 1.15mno 0.91t 0.72u 0.56v 0.59v 0.62v 0.85e Neem 3% 1.42h 1.26jk 1.17lmn 1.05pq 0.91t 0.93st 0.95rst 1.10d Trichogramma 1.45gh 1.31ij 1.25jkl 1.15mno 1.01qrs 1.03pqr 1.07opq 1.18c Neem 2% 1.51g 1.42h 1.31ij 1.22klm 1.11nop 1.15mno 1.17lmn 1.27b Control 3.42f 3.57e 3.71d 3.84c 3.89c 3.99b 4.11a 3.79a Mean 1.83a 1.74b 1.67c 1.60d 1.50f 1.54e 1.58d *Means in rows and columns followed by different letters are significantly different at p-value ≤ 0.05 LSD value (P ≤ 0.05) for treatment = 0.033 LSD value (P ≤ 0.05) for time = 0.039 LSD value (P ≤ 0.05) for treatment× time intervals = 0.088 Statistical analysis of the data revealed that both botanical, commercial insecticides and Trichogramma significantly (P ≤ 0.05 and F-value = 73.24) reduced the larval population of H. armigera. Lowest larval population (0.85 larvae/plant) was recorded in the plots treated with commercial insecticide Radiant, followed by larval population (1.10 larvae/plant) was recorded in the plots treated with botanical insecticide Neem (3%). The larval population (1.18 larvae/plant) was noted in the plots treated with biological agent (Trichogramma). Highest larval population (3.79 larvae/plant) was observed in control treatment. Different time intervals also significantly (P ≤ 0.05 and F-value = 10652.0) reduced the larval population of H. armigera. Lowest larval population (1.50 larvae/plant) was recorded after 7 days, followed by the larval population (1.54 larvae/plant) was recorded after 10 days. Highest larval population (1.83 larvae/plant) was recorded after 24 hours. Page 13/19 Interaction effect of time interval and treatments were also found significant (P ≤ 0.05 and F-value = 44.01). Lowest larval population (0.56 larvae/plant) was recorded after 7 days in the plots treated with commercial insecticide Radiant, followed by the larval population (0.59 larvae/plant) was recorded after 10 days in the plots treated with Radiant. Highest larval population (4.11 larvae/plant) was recorded after 15 days in control plots. Effect on percent fruit damage A gradational decline was found in percent fruit damage by the application of botanical, commercial insecticides and Trichogramma. Effect of each treatment on percent fruit damage by number and weight was recorded and presented in Table 7 during the experiment. Table 7 Effect of each treatment on damage of tomato Treatments % damaged fruits (No.) % decrease over control (No.) % damaged fruits (weight) % decrease over control (weight) Radiant 6.13 e 83.03 5.53 e 85.30 Neem 3% 9.76 d 72.98 8.11 d 78.45 Trichogramma 11.53 c 68.08 12.15 c 67.71 Neem 2% 14.42 b 60.08 15.67 b 58.36 Control 36.12 a 0.00 37.63 a 0.00 LSD value 0.70 - 0.40 - *Means followed by different letters are significantly different at p-value ≤ 0.05 1. Percent fruit damage by number Analysis of variance of data showed that spray application of botanical, commercial insecticides and Trichogramma significantly (P ≤ 0.05 and F-value = 11094.4) reduced percent tomato fruit damage by number. Highest reduction of fruit damage by number (6.13) with 83% decrease over control was recorded in the plots treated with commercial insecticide Radiant, followed by fruit damage by number (9.76) with 73% decrease over control was recorded in the plots treated with botanical insecticide Neem (3%). Reduction of fruit damage (11.53) with 68% decrease over control was noted in the plots treated with Trichogramma. Lowest reduction of fruit damage (36.12) was observed in control plots. Commercial insecticide Radiant was found more effective in reduction of fruit damage by number compared to botanical insecticide (Neem 2 and 3%) and Trichogramma chilonis. 2. Percent fruit damage by weight Page 14/19 Effect of spray application of botanical, commercial insecticides and Trichogramma chilonis on percent tomato fruit damage by weight was also found significant (P ≤ 0.05 and F-value = 3058.00). Highest fruit damage by weight (37.63) was recorded in control. Lowest fruit damage by weight (5.53) with 85% decrease over control was recorded in the plots treated with commercial insecticide Radiant, followed by fruit damage by weight (8.11) with 78% decrease over control was recorded in the plots treated with botanical insecticide Neem (3%). Reduction of fruit damage by weight (12.15) with 68% decrease over control was noted in the plots treated with T. chilonis. Results clearly indicated that strong positive reduction in percent fruit damage by weight was found in the plots treated with commercial insecticide Radiant compared to all other treatments. Effect of each treatment on average yield of tomato (kg) The data recorded on average yield per plot and total marketable yield of tomato are presented in Table 8. Table 8 Effect of botanical, commercial insecticides and Trichogramma on average yield of tomato (kg) recorded per plot and per hectare during 2019 Treatments Yield (kg) plot− Total marketable yield (kg ha− 1) Percent increase over control Radiant 11.5 a 9583 a 79.69 Neem 3% 10.1 b 8417 b 57.83 Trichogramma 9.1 c 7583 c 42.19 Neem 2% 8.6 d 7167 d 34.39 Control 6.4 e 5333 e 0.00 LSD value 0.40 334.47 - 1 *Means followed by different letters are significantly different at P-value ≤ 0.05. 1. Yield per plot (kg) Statistical analysis of the data indicated that spray application of botanical, commercial insecticides and Trichogramma chilonis significantly (P ≤ 0.05 and F-value = 235.58) increased tomato yield per plot. Maximum tomato yield (11.5 kg plot− 1) was recorded in the plots treated with commercial insecticide Radiant, followed by the tomato yield (10.1 kg plot− 1) was recorded in the plots treated with botanical insecticide Neem (3%). Tomato yield of (9.1 kg plot− 1) was noted in the plots treated with Trichogramma. Minimum tomato yield (6.4 kg plot− 1) was observed in control plots. 2. Total marketable yield (kg ha) Page 15/19 Analysis of variance of the data revealed that total marketable yield of tomato was also significantly (P ≤ 0.05 and F-value = 235.86) affected by spray application of botanical, commercial insecticides and Trichogramma chilonis. Highest tomato total marketable yield (9583 kg ha− 1) with 80% increase over control was recorded in the plots treated with commercial insecticide Radiant, followed by the tomato total marketable yield (8417 kg ha− 1) with 58% increase over control was recorded in the plots treated with botanical insecticide Neem (3%). Tomato total marketable yield (7583 kg ha− 1) with 42% increase over control was recorded in the plots treated with T. chilonis. Lowest tomato total marketable yield (5333 kg ha− 1) was observed in control plots. Table 9 Net income per hectare Treatments Cost of treatments Cost of labor, irrigitaion (Rs) Total cost Total income Net CBR income Radiant 10800 3000 13800 191660 177860 (13.89) Neem 3% 12960 3000 15960 168340 152380 (10.55) Trichocards 4500 3000 7500 151660 144160 (20.22) Neem 2% 8641 3000 11641 143340 131699 (12.31) Control - - - - - Average price of tomato per kg = Rs. 20; Neem oil per liter = Rs. 1200, Radiant 100 ml/hectare = Rs. 3600, cost incurred on trichocards = 1500 per application, cost of spray @ 3000 per spray/hectare 3. Cost benefit ratio analysis of the treatments Economic analysis of the data for efficacy of botanical extracts (Neem 2 and 3%) commercial insecticide Radiant, trichocards for the control of tomato fruit borer H. armigera has been presented in Table 9. The highest benefit-cost ratio (BCR) of 20.22 was recorded in Trichogramma fallowed by the Radiant which produced BCR of 13.89, next to this treatment. Botanical insecticide (Neem 2%) also gave comparable BCR of 12.31, whereas Neem 3% gave lowest BCR value of 10.55. DISCUSSION Fruit borer Helicoverpa armigera is an important insect pest of tomato in Pakistan. The pest infestation decrease the average tomato yield/ha and also make it unfit for human consumption. Therefore, it's on time and safe management is essential. The present research investigated the comparative field efficacy of some treatments used against tomato fruit borer. Results showed that among different treatments the lowest pest infestation was recorded in chemical treated plots (Radiant). These results are comparable to Page 16/19 those of some earlier researchers; Babar et al. (2016) recorded equal effect of Radiant against H. armigera as our experiment demonstrates. It was recorded 73% decline in H. armigera larvae infestation in the plots treated with Radiant and concluded that Radiant prove to be the best against tomato fruit borer. Sohail et al. (2004) experimented different chemicals to control the population density of H. armigera. Results depicted that cypermethrin can effectively control the pest and aid in greater yield. Ghosal et al. (2012) applied different chemicals in a tomato field against H. armigera and observed that chemical control effectively reduce the infestation of H. armigera in the field. In case of egg infestation the commercial insecticide Radiant gives the best result in comparison with botanical extracts (Neem 2 and 3%) and bio agent (Trichogramma chilonis). Radiant proved the maximum ovicidal effect against the H.armigera eggs followed by Neem 3% and T. chilonis in the reduction of mean number of eggs/plant. Neem 2% showed the lowest ovicidal effect against the H. armigera eggs while the maximum eggs infestation was recorded in the control plot. Our results are same with Rizvi and Jaffar (2015) who applied combination of pesticides and plant extracts against H. armigera in tomato field. They found that application of insecticides and plant extracts greatly suppress the density of H. armigera and increase the production capacity resulting in the wellbeing of farmer. Abbas et al. (2015) examined the infestation variance of H. armigera on different crops and reported that H. armigera is destructive pest that can result in significant yield losses. To minimize losses from H. armigera he suggested that bio-control agents and chemical control should be used. They also reported that radiant is the most appropriate insecticide because of its effectiveness in minimizing H. armigera infestation and less persistent in the environment as compared to the other insecticides. Similar results were reported by Visnupriya and Muthukrishnan (2017) who revealed that spinetoram (radiant) has short residual period of 11 days in reference to other treatments. In case of percent fruit damage by weight and number, the lowest tomato fruit damage was reported in the plots treated with commercial insecticide radiant. In botanical insecticides minimum fruit damage was recorded in the plots treated with Neem 3% followed by the T. chilonis. The highest percent fruit damage was observed in control plots. The results are in similarity with Rasheed et al. (2019) who reported that maximum numbers of fruits were damaged in control plots while the lowest number fruits were damaged in the plots treated with radiant. They also reported that plots with lowest larval infestation and less number of damaged fruit results in higher marketable yield. As larvae of H. armigera bore into the tomato fruit, eventually the fruit become unhealthy for consumption and losses its value in market. Our results are also comparable with Usman et al. (2018) who reported that higher infestation of larvae results in lower yield. In case of yield the maximum tomato fruit yield was recorded in the plots treated with commercial insecticide Radiant, followed by the fruit yield obtained from the plots treated with botanical extract Neem 3% and bio agent (T. chilonis). Minimum tomato fruit yield was noted in control plots. Our results are in coherence with Utti (2017) who reported that with application of insecticides effectively increase the yield. Kumar et al. (2013) also revealed that management of field with various insecticides can enhance the Page 17/19 production of tomato. Mahla et al. (2017) also reported same impact from application of insecticides with low infestation and high marketable yield from treated plots. In case of CBR values recorded our results are same with Abbas et al. (2020) and Ullah et al. (2012) who reported the effectiveness of T. chilonis against this pest and found similar results. Trichocards do not need any special equipment or skilled person as well as very economical for application. CONCLUSION AND RECOMMENDATIONS 1. It is concluded from the results of the experiment that Radiant (Spinetoram), Trichocards and Neem 3% showed higher larval and eggs population suppression compared to control treatment. Radiant proved best in reduction of larval and eggs population of H. armigera. 2. Application of Radiant (Spinetoram), Trichocards and Neem 3% also significantly reduced tomato fruit damage which as a result increased total marketable yield of tomato compared to control. 3. However, bio control treatment incurred low cost of control which resulted with high cost benefit ratio. 4. It was concluded from the current research that Radiant (Spinetoram), Trichocards and Neem 3% comparatively have a maximum potential in reduction of fruit infestation which as a results gives higher yield with high percentage of marketable fruits and with highest cost benefit ratio. It is recommended that that all these effective treatments could be integrated against pest control program of H. armigera in tomato crop. Declarations Authors Contribution: Amani Mulk Khan conduct the experiment, Ahmad-Ur-Rahman Saljoqi designed the experiment, technical and language checked, Mehran Ullah analyzed data and wrote the manuscript. The authors have no financial or proprietary interests in any material discussed in this article. References 1. Abbas S, Tahir M.J, Naeem A, Sarfraz AS, Rafiq M, Ahmad MS (2015) Effectiveness of chlorantraniliprole, indoxacarb, fluendiamide, spintoram, thiamethaxim, emamectin benzoate and lufenuron in Pakistan environment. J Phyto 43:577-583. 2. Amalendu G, Chatterjee M, Roy A (2009) Bio-efficacy of spinosad against tomato fruit borer (Helicoverpa armigera) and its natural enemies. J Hort Fore 2(5):108-111. 3. Atwal AS (1976) Agricultural Pests of India and South East Asia. Kalyani Publishers New Delhi., Pp. 528. 4. Ayvaz A, Karasu E, Karaborklu S, Yilmaz S (2008) Dispersal ability and parsitizition performance of egg parasitoid Trichogramma evanescene Westwood in field and storage conditions. Turk J Biol (32): 27-33. Page 18/19 5. Babar TK, Hasnain M, Aslam A, Ali Q, Ahmad KJ, Ahmad A. Shahid M (2016) Comparative bio efficacy of newer insecticides against tomato fruit borer, Helicoverpa armigera (Hubner) on tomato crop under field conditions. Pak Entomol 38(2):115-122. 6. Baloch FA (1994) Vegetable crops. In: Horticulture. National Book Foundation, Islamabad. Pp. 508. 7. Ghosal A, Chatterjee ML, Manna D (2012) Studies on some insecticides with novel mode of action for the management of tomato fruit borer (Helicoverpa armigera Hub.). J Crop weed 8(2):126-129. 8. Hassan SA (1992) Guideline of side effect of plant protection product on Trichogramma. chilonis. In guideline for testing effect of pesticides on beneficial organism. Bull 105(3):19-39. 9. Hoffmann H, Hadie D, Burt J (2007) Tomato pests in the home garden and their control. Dept Agric Austr Gard No. 34:82-88. 10. Husain M (1993) The control of rice borer in Bangladesh. 8(8): 28-30. 11. Hussin B, Bilal S (2007) Efficacy of different insecticides on tomato fruit borer Helicoverpa. armigera. J Entomol 4(1):64-67. 12. Kumar V, Kakkar G, McKenzie CL, Seal DR, Osborne LS (2013) An overview of chilli thrips, Scirtothrips dorsalis (Thysanoptera: Thripidae) biology, distribution and management. Weed and pest controlConventional and new challenges. Pp. 53-77. 13. Mahla M, Lekha K, Singh V, Swami H, Choudhary RS (2017) Efficacy of different insecticides against pest complex of tomato and effect on their natural enemies. J Entomol Zool Stud 5(5):229-234. 14. Rasheed I, Shah SF, Sarwar J, Usman A, Shah M, Usman M, Amin F, Nisar N (2019) Screening of different insecticides against Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) and its effect on yield of tomato crop. Pure Appl Biol 8(1):496-502. 15. Rizvi SAH, Jaffar S (2015) Efficacy of some selected chemical insecticides and bio-pesticides against Helicoverpa armigera under the agro climatic condition of Gilgit Baltistan. Pak J Entomol Zool 3(4):50- 52. 16. Shaheen N (2008) Is organic forming suitable solution for Pakistan. SDPI Res and News Bull 15(2):78-81. 17. Sohail A, Khurram Z, Naeem RS (2004) Validation of chemical control of gram pod borer, Helicoverpa armigera with new insecticides. Ind J Plant Prot 4:41-49. 18. Usman A, Ali MI, Shah M, Amin F, Sarwar J (2018) Comparative efficacy of indigenous plant extracts and a synthetic insecticide for the management of tomato fruit worm (Helicoverpa armigera Hub.) and their effect on natural enemies in tomato crop. Pure Appl Biol 7(3):1014-1020. 19. Utti PS (2017) Bio-efficacy of newer insecticides against fruit borer (Helicoverpa armigera hub.) of tomato. M.Sc (Hons) Thesis. Dept. of Entomol, Coll. Agri. Indore, pp. 51. 20. Visnupriya M, Muthukrishnan N (2017) Persistence toxicity and field evaluation of green insecticide spinetoram 12 sc w/v (11.7% w/w) against Helicoverpa armigera hubner on okra. Int. J. Curr. Microbiol. App Sci 6(11):2547-2555. Page 19/19 

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Biosystems Diversity ISSN 2519-8513 (Print) ISSN 2520-2529 (Online) Biosyst. Divers., 25(4), 354–360 doi: 10.15421/011752 Species-specific morphological characteristics of adult and embryonic Capillariа obsignata roundworms (Nematoda, Capillariidae) V. A. Yevstafyeva*, V. V. Stybel**, T. A. Sharavara*, V. V. Melnychuk*, I. О. Yasnolob*, А. А. Antipov***, V. P. Goncharenko***, T. I. Bakhur*** *Poltava State Agrarian Academy, Poltava, Ukraine **Lviv National University of Veterinary Medicine and Biotechnologies named after S. Z. Gzhytskyj, Lviv, Ukraine ***Bila Tserkva National Agrarian University, Bila Tserkva, Ukraine Article info Received 12.10.2017 Received in revised form 17.11.2017 Accepted 19.11.2017 Poltava State Agrarian Academy, Skovorody Str., 1/3, Poltava, 36003, Ukraine. Tel.:+38-050-183-78-78. E-mail: evstva@ukr.net Lviv National University of Veterinary Medicine and Biotechnologies named after S. Z. Gzhytskyj, Pekarska Str., 50, Lviv, 79000, Ukraine. E-mail: vstybel@ukr.net Bila Tserkva National Agrarian University, Soborna Str., 8/1, Bila Tserkva, 09100, Ukraine. E-mail: antipov_anatolii@ukr.net Yevstafyeva, V. A., Stybel, V. V., Sharavara, T. A., Melnychuk, V. V., Yasnolob, I. О., Antipov, А. А., Goncharenko, V. P., & Bakhur, T. I. (2017). Species-specific morphological characteristics of adult and embryonic Capillariа obsignata roundworms (Nematoda, Capillariidae). Biosystems Diversity, 25(4), 354–360. doi:10.15421/011752 The abundance of Capillaria obsignata Madsen, 1945, the causative agent of chicken capillariasis, was studied in the central region of Ukraine. Differential morphometric characters are determined for adult males and females of C. obsignata. The species-specific variability of its morphological parameters is characterized. New data is received on the meristic characters of C. obsignata eggs extracted from different substrates. The stages of embryonic development are determined in laboratory cultures of these nematodes, taking into account their meristic and morphological changes. Capillaria obsignata parasites are very common at the poultry farms of Poltava and Kyiv regions of Ukraine. Their abundance index is 3.4, and the intensity of infection varies from 3 to 317 specimens. Differential morphometric species-specific characters of adult C. obsignata males are the shape and size of the pseudobursa, spicule, spicule sheath. Adult females of the species can be identified by the shape and size of the vulva, the distance from the esophagus end to the vulva, and the length of the vagina. The meristic characters of nematode eggs, though typical for the species, may change depending on the sampled substrate. The parasite’s embryonic development involves five morphologically and meristically different stages. The invasive larvae of this species developed in the eggs before the 12th day of culturing in laboratory conditions at 25 °С. The invasive larvae’s viability was 90.3 ± 1.5%. Keywords: chicken; abundance index; adult roundworm; nematode eggs; differential characters Introduction Parasitic nematodes are one of the most numerous and widespread groups of parasitic worms. The suborder Trichurata Skijabin et Schulz, 1928 is an especially prominent group of parasitic roundworms, including capillariid nematodes (Capillariidae NeveuLemaire, 1936). The taxonomy and taxonomy of capillariids are contentious. Different capillariid species parasitize humans, monkeys, wild and domestic carnivores, rodents, fish, reptiles, ruminants, and wild and domestic poultry. The results of morphological studies are of great importance in the consideration of systematics, developmental biology and phylogeny of capillariids (Petrochenko and Kotelnikov, 1976; Lomakin and Romashov, 1987; Moravec et al., 1994; Timi et al., 2006; Moravec and Muzzall, 2009). The wide distribution of Capillaria obsignata Madsen, 1945 which parasitizes both poultry (Gallus gallus dom., Meleagris gallopavo) and wild birds (Columba livia, Zenaidura carolinensis, Perdix perdix, Phasianus colchicus, Sturnus vulgaris) in areas with varying climatic conditions is confirmed in numerous studies (Wakelin, 1965, 1966; Park and Shin, 2010; Gürler et al., 2012; Pasechnik, 2013). This adaptation to parasitizing different avian species in various geoclimatic regions, and its prevalence over other capillariid species are sustained by this roundworm’s life cycle. It does not require an intermediate host, allowing fast distribution and adaptation of C. оbsignata, especially among chickens. This species was found at poultry farms in Africa (Mukaratirwa et al., 2001; Mukaratirwa and 354 Khumalo, 2010), Vietnam (Schou et al., 2007), Morocco (Hassouni and Belghyti, 2006), Germany (Kaufmann et al., 2011), and USA (Yazwinski et al., 2013). The ratio of infected chickens ranged from 0.3% to 75.3%, and the intensity of infection was up to 1280 nematodes. Many researchers point out the morphological taxonomic characters of C. obsignata and other avian capillariid species, such as: posterior end of male, spicule, ornamentation of spicule sheath, the shape and number of stichocytes (large glandular cells along the posterior part of the esophagus), and the shape and size of eggs, and the vulva area in females (Paramonov, 1957; Gagarin, 1971; Tanveer et al., 2013). Lately it has been proposed to consider capillariid morphometry characteristics, since several species are difficult to identify because of their morphological similarity both in males and females. The reviewed characters are the length and width of body, the anterior to posterior body portions length ratio, in males – the length and width of spicule and in females the distance between the esophagus end and the vulva, body width at the vulva area, and the distance between the anus and posterior end (Kajerová and Baruš, 2005; D’ávila et al., 2012, 2017). Also, studies indicate that the shape, size and the ornamentation of the egg’s outer surface are species-specific characters for the family Capillarіidae. They are divided by the shape into three major groups: elongated (oblong, cylindrical), oval (rounder, barrel-shaped), and round (lemonshaped) (Nasirov, 1981; Romashov, 1985). There is still a lot of confusion regarding the systematic placing and the species composition of avian capillariasis pathogens. For Biosyst. Divers., 25(4) example, Capillaria obsignata is also referred to as Baruscapillaria columbae, Baruscapillaria obsignata, and Capillaria columbae (Levine, 1938; Wehr, 1939; Tamaru et al., 2015). Lacking a system of well-defined diagnostic tests based on evaluation of qualitative and quantitative morphological characters, it is quite difficult to identify capillariid species. In Ukraine, the prevalence of C. obsignata in poultry has been studied but irregularly, and the worm’s morphometric characteristics of adult and embryonic stages are insufficiently reviewed. The purpose of this article was to find out the prevalence of Capillaria obsignata (Madsen, 1945) in chickens (Gallus gallus dom.) in the central region of Ukraine, and also to determine the differential taxonomic characters of adult and embryonic stages of this species, taking into account their biology. Materials and methods The studies were conducted in 2015–2017 at the Laboratory of Parasitology and Veterinary-Sanitary Expertise of the Department of Veterinary Medicine of Poltava State Agrarian Academy, and in the I. I. Schmalhausen Institute of Zoology, National Academy of Sciences of Ukraine. The population structure of C. obsignata was studied in poultry farms of Poltava and Kyiv regions. The main indicators were the abundance index and the intensity of infection (Ripolovskyj and Yuskiv, 2010). Helminthes were collected during the complete helminthological dissection of the digestive tract (mouth, crop, esophagus, small intestine and colon) of dead or killed chickens (Skrjabin, 1928). The roundworms were identified according to (Skrjabin, 1957). In total, 1,434 adult specimens of C. obsignata nematodes were studied, 1,273 of them female and 161 male. To study the morphological and meristic parameters of the roundworms’ eggs, they were extracted from various substrates: the female nematode gonads (Eg) and the feces of infected chickens (Ef). The studied characters were the shape, surface, length and width of eggs, egg plug length and width, eggshell thickness, and the areas of the outer and inner egg surfaces. The embryogenesis of C. obsignata was studied in eggs extracted from the feces of infected chickens and cultured to the invasive stage in a thermostat at 25 °С for 10 to 12 days. Every two days the culture was examined under a microscope. The experiments were conducted in triplicate. Morphometric parameters of embryonic and adult stages of C. obsignata were analyzed using ImageJ for Windows® (version 2.00) in interactive mode using ×10 and ×40 objective, and ×10 photo eyepiece. To calibrate the image analyzer, the ruled scale of an ocular micrometer was calibrated with the scale of stage micrometer included in MikroMed microscope kit. Microphotography was performed using a 5 Mpix digital camera of MikroMed (China) microscope. The material and significance levels were analyzed using standard methods of statistical processing. All the data are reported as the sample mean ± the standard deviation (SD) (Lapach et al., 2001). distinctive visual signs. This study confirmed the sexual dimorphism in this species (Table 1). The female Capillaria is on average 14.75 ± 0.82 mm, 23.7% longer than the male (11.25 ± 0.63 mm). The females are also wider by 10.2−17.2% than males. The microscopy study revealed no distinctive morphologic characters of the anterior end in males and females (Fig. 1). In males, the posterior end structure in the area of the sexual bursa is the typical morphological character of the species. In C. obsignata it is weakly developed as a pseudobursa. The pseudobursa is not large (25.45 ± 1.54 μm in length, 35.54 ± 1.42 μm in width), transparent and not lobed, and on each side it has a wide rounded ray, which slightly narrows at the base (Fig. 2а). The distance between rays is 13.57 ± 0.74 μm. A mature C. obsignata male has only one spicule, thin and long (1.62 ± 0.07 mm) with thin rounded distal end (5.67 ± 0.39 μm) (Fig. 3а), which is 3.8 times thinner than the proximal end (21.45 ± 1,72 μm). The latter has a funnelshaped, 27.15 ± 0.34 μm long dilatation (Fig. 3b). The spicule is covered by a thin sheath, transversely striated and lacking spikes (Fig. 2b). The average length of the spicule sheath is 2.80 ± 0.41 mm. It is 1.4 times wider at the proximal end (23.24 ± 1.20 μm) than at the distal end (16.89 ± 0.40 μm). Fig. 1. Anterior end of Capillaria obsignata Results It was established that C. obsignata nematodes are quite common in the central region of Ukraine. The dissection of 425 chickens resulted in 1,434 specimens of C. obsignata roundworms, and the intensity of infection ranged from 3 to 317 specimens. The average abundance index of this parasite was 3.4. The females were found much more often (1,237) than males (161), the corresponding ratio is 7.9 to 1.0. It was also determined that 3.5% of the studied chickens had only female roundworms, supporting the need for identification characters for female nematodes. It is found that C. obsignata nematodes have several distinguishing morphological parameters which facilitate the species identification. Thus, the capillariid body is filiform both in males and in females, barely visible to the naked eye, translucent, without any b Fig. 2. Posterior end of ♂ Capillaria obsignata: а – pseudobursa with rays, b – spicule sheath (spicule is extruded) Biosyst. Divers., 25(4) 355 A mature C. obsignata female has such species-specific morphological characters as vulval area structure and meristic indexes of body in the area of sexual organs. The vulva is a slit with cuticle protrusion in the shape of a genital or vulval lip. The latter is quite unstable in size: its length varies from 22.9 to 29.7 μm, and its height from 2.0 to 7.2 μm (Fig. 4). Table 1 Morphometric parameters of adult Capillaria obsignata roundworms (n = 10) ♂ Characters Body length, mm Body width at: – anterior end, μm – esophagus end, μm – posterior end, μm – vulva, μm – anus, μm Distance from esophagus end to vulva, μm Length of cuticle protrusion in the vulva area, μm Height of cuticle protrusion in the vulva area, μm Length of vagina, μm Length of spicule, mm Length of funnel-shaped dilatation of spicule, μm Width of spicule at: – proximal end, μm – after dilatation, μm – in the middle, μm – distal end, μm Length of spicule sheath, mm Width of proximal end of spicule sheath, μm Width of distal end of spicule sheath, μm Length of pseudobursa, μm Width of pseudobursa, μm Width of pseudobursal base, μm Distance between pseudobursal rays, μm ♀ M ± SD 11.25 ± 0.63 min – max 10.5–12.5 M ± SD 14.75 ± 0.82 min – max 13.5–16.0 8.47 ± 0.56 47.27 ± 1.94 38.48 ± 1.78 – – – – – – 1.62 ± 0.07 27.15 ± 1.06 7.4–9.4 44.1–49.6 35,2–41,3 – – – – – – 1.5–1.7 25.7–28.9 9.43 ± 0.53 57.09 ± 2.65 – 55.26 ± 2.70 34.59 ± 1.90 82.65 ± 4.63 27.39 ± 2.41 4.46 ± 1.77 173.66 ± 9.81 – – 8.3–10.2 52.1–61.3 – 50.7–59.1 31.9–37.3 76.6–91.3 22.9–29.7 2.0–7.2 160.7–185.6 – – 21.45 ± 1,72 11.73 ± 0,94 8.93 ± 0.57 5.67 ± 0.39 2.80 ± 0.41 23.24 ± 1.20 16.89 ± 0.40 25.45 ± 1.54 35.54 ± 1.42 33.70 ± 2.07 13.57 ± 0.74 18.7–24.0 10.3–13.2 8.2–9.7 5.1–6.2 2.2–3.6 21.1–24.5 16.0–17.5 23.6–28.1 33.1–37.5 30.2–37.5 12.3–14.8 – – – – – – – – – – – – – – – – – – – – – – Note: “–“ – рarameters were not defined. а b Fig. 3. The spicule of ♂ Capillaria obsignata: а – proximal end, b – distal end Fig. 4. Vulval area of ♀ Capillaria obsignata: the variability of cuticle lip size The differential character is the distance between the esophagus end and the vulva, which is 82.65 ± 4.63 μm on average. The vulva is located behind the junction of the esophagus with the intestine. The vagina has a well developed muscular wall and is 173.66 ± 9.81 μm long. The uterus is filled with eggs. The tail end of the female is blunt, the anus is subterminal, the body width in this area is 34.59 ± 1.90 μm. 356 The outer structure of C. obsignata eggs is typical for Capillariidae: barrel-shaped, with egg plugs on both ends, of various brown shades. The egg has a thick transparent eggshell and is not mature (Fig. 5). Comparison of C. obsignata eggs extracted from different substrates reveals a significant difference by all considered morphometric parameters (Table 2). Biosyst. Divers., 25(4) day the cleaving embryos reached 66.3% of all. Later, the number of C. obsignata eggs, in which the cleavage process was occurring, decreased to 22.0% on day 6, and then to 2.3% on day 8. The formation of bean-like embryo occurred in days 4 to 6. а а b Fig. 5. The eggs of Capillaria obsignata extracted from different substrates: а – from female nematode gonads, b – from feces of infected chickens b Table 2 Meristic parameters of Capillaria obsignata, eggs extracted from different substrates (n = 10) Indicators Total egg length, μm Length of egg without the egg plugs, μm Egg width, μm Eggshell thickness, μm Inner surface area of the egg, μm2 Outer surface area of the egg, μm2 Eg Ef Eg Ef Eg Ef Eg Ef Eg Ef Eg Ef min 50.5 60.1 45.3 58.3 23.9 24.5 2.1 2.9 631.9 782.4 642.0 785.7 max 56.2 74.6 49.6 70.1 27.0 32.4 2.9 3.6 687.4 847.3 691.1 855.1 М ± SD 53.1 ± 2.1 65.3 ± 5.1*** 47.2 ± 1.3 63.0 ± 4.4*** 25.6 ± 1.1 28.4 ± 2.4** 2.39 ± 0.28 3.22 ± 0.26*** 661 ± 16 811 ± 19*** 666 ± 14 819 ± 19*** c Note: Eg – Capillaria eggs extracted from female nematode gonads, Ef – Capillaria eggs extracted from feces of infected chickens; ** – Р < 0.01; *** – Р < 0.001 – compared to the Eg parameter values. Hence, nematode eggs from the feces of infected chickens are longer by 18.7–25.1% (Р < 0.001) than the eggs extracted from female nematode gonads if either the total egg length or egg length without the egg plugs are analyzed. Simultaneously, the eggs from chicken feces are wider by 10.1% (Р < 0.01), and the eggshell thicker by 25.8% (Р < 0.001) compared to eggs from nematode gonads. Also, the surface areas of C. obsignata eggs are very differrent. The eggs from chicken feces have larger outer and inner surface areas, by 18.5% and 18.6% respectively (Р < 0.001) than the eggs from nematode gonads. It is established that the embryogenesis of C. obsignata at 25 °С in the laboratory culture occurs in 12 days and can be divided in five provisory stages: zygote (Fig. 6а), morula or embryo cleavage (Fig. 6b), bean-like embryo (Fig. 6c), tadpole embryo (Fig. 6d), invasive egg (larva, Fig. 6e). The viability of C. obsignata eggs is quite high during the embryogenesis (90.3 ± 1.5%, Table 3). At the beginning, 100% of Capillaria egg culture was at the zygote stage. From the second to the fourth days of culture, the eggs started to change with the cleavage of the embryo and the formation of morula. Thus on day 2, 11.3% eggs were cleaving, on the 4th d e Fig. 6. Stages of embryonic development of Capillaria obsignata: а – zygote; b – morula; с – bean-like embryo; d – tadpole embryo; e – invasive larva Then, from 15.6% to 49.6% of eggs were at this stage of development. The formation of tadpole embryo was recorded at the days 8 to 10 of embryogenesis. At the eighth day, 71.6% of eggs were at this stage, and on the 10th day this number decreased to Biosyst. Divers., 25(4) 357 4.0%. Larval development occurred gradually since the eighth day of culture when only 7.0% of eggs were invasive. At the 10th day, 84.3% of all eggs became invasive. At the 12th day, the maximum number of invasive eggs was observed, 90.3%. The cessation of development and subsequent death was recorded in 9.6% of C. obsignata eggs. The stages of embryonic development of C. obsignata eggs were typical and their morphological changes were supported by meristic parameters (Table 4). Table 3 The embryonic development of Capillaria obsignata in experimental culture (М ± SD, n = 100) Day of culture Before culturing 2 4 6 8 10 12 zygote 100 88.66 ± 2.52 18.00 ± 2.00 9.66 ± 1.53 – – – morula (cleavage of embryo) – 11.33 ± 2.52 66.33 ± 3.79 22.00 ± 2.00 2.33 ± 1.53 – – Stages of egg development, % development of bean-like embryo tadpole embryo – – – – 15.66 ± 2.08 – 49.66 ± 2.51 18.67 ± 3.05 9.33 ± 2.51 71.66 ± 2.08 2.00 ± 1.00 4.00 ± 1.00 – – larva – – – – 7.00 ± 3.61 84.33 ± 3.05 90.33 ± 1.53 Cessation of development – – – – 9.66 ± 1.53 9.66 ± 1.53 9.66 ± 1.53 Table 4 Meristic parameters of Capillaria obsignata embryonic development in experimental culture (М ± SD, n = 10) Parameters, μm Length Width Eggshell thickness Before cultivation 65.34 ± 5.12 28.42 ± 2.35 3.14 ± 0.24 2 67.88 ± 4.91 28.87 ± 2.05 3.12 ± 0.25 4 69.09 ± 5.21 29.14 ± 2.02 3.11 ± 0.25 Day of culture 6 8 71.08 ± 6.28* 72.27 ± 4.32** 29.24 ± 2.97 29.57 ± 2.57 3.09 ± 0.26 3.09 ± 0.29 10 72.66 ± 3.69** 30.61 ± 1.67* 3.08 ± 0.29 12 73.57 ± 7.53** 30.72 ± 2.56* 3.08 ± 0.28 Note: * – Р < 0.05, ** – Р < 0.01 compared to parameters before cultivation. Thus, in embryonic development of bean-like embryo, the egg length significantly increased, starting at the sixth day of cultivation, by 4.5% (Р < 0.05). At the eighth day, the embryogenesis was at the stage of tadpole embryo, and its length increased by 6.1% (Р < 0.05) compared to before cultivation. At the 10th and 12th days, the length of invasive larva increased still by 10.1% and 11.2% (Р < 0.01) respectively. The egg width began to change at the larval formation stage, and increased by 7.5% (Р < 0.05) at the 12th day compared to that at the zygote stage. The eggshell thickness decreased but insignificantly. Thus, C. obsignata eggs differ depending on the stage of embryogenesis and the initial substrate. These specifics should be considered in species identification. Discussion The population of C. obsignata is well adapted to parasitizing chickens (Gallus gallus dom.) in the climatic conditions of central Ukraine (Poltava and Kiev regions), which is confirmed by the population statistics. The abundance index is 3.4, the intensity of capillariasis infection is 317 specimens in several cases. It is well known that C. obsignata is adapted to parasitizing domestic, wild, and synanthropic birds. The occurrence of cross infection means that this nematode is not a species-specific parasite (Levine, 1938; Wakelin, 1965; Gürler et al., 2012). This factor facilitates the wide distribution of C. obsignata in many countries, as confirmed by many studies (Permin, 1999; Mukaratirwa and Khumalo, 2010; Kaufmann et al., 2011; Yazwinski et al., 2013). In Ukraine, there are a few studies of the abundance of C. obsignata in birds, revealing the significant infection of chickens, especially floor-reared (extensiveness of infection up to 100%) in Dnepropetrovsk region (Zaikina and Marshalkina, 2015). We found out that the studied population of C. obsignata is represented mostly by females (88.8%), and 3.5% of studied birds had no male parasites. This supports previous findings (D’ávila et al., 2012) which also testifies to the roundworm’s biological adaptation to certain conditions and interaction with the host. That is, the females live longer to provide successful reproduction, and the males leave the host after fertilization. New data are found on the morphometric characteristics of adult and embryonic stages of C. obsignata development. The species has been studied for a significant period, and there are widely known identification keys (Skrjabin, 1957; Ivashkin et al., 1971). However, the keys do not incorporate the specific variability which 358 can affect both the morphological and meristic parameters of these roundworms. The average length of mature females is 14.75 ± 0.82 mm, their width in various body parts ranges from 9.43 ± 0.53 to 57.09 ± 2,65 μm. These values are lower in males by 10.2–23.7%, their length is 11.25 ± 0.63 mm, their width is 8.47 ± 0.56 to 47.27 ± 1,94 μm. However, several sources give the length of C. obsignata females from 9.1 to 40.0 mm, and the length of male roundworms from 5.83 to 18.6 mm (Kajerová and Baruš, 2005; D’Ávila et al., 2017). The mature females and males in our study were unequal by their body lengths (♀ 13.5 to 16.0 mm, ♂ 10.5 to 12.6 mm). This, too, confirms their adaptive morphological variability, which causes the difference in the nematode sizes. Among other typical meristic parameters, there is the length of the pseudobursa (35.54 ± 1,42 μm), distance between the rays of the pseudobursa (13.57 ± 0.74 μm), and the lengths and widths of the spicule and spicule sheath at the distal and proximal ends. All this should be taken into account in species identification. In most of the identification keys, the slit vulva and insignificant vulval lip are thought to be typical for C. оbsignata females. We, however, must point out that the size and height of this vulval lip are very variable (2.0 to 7.2 μm), thus making difficult the species identification. Hence, the meristic characteristics of females should also be considered, such as the distance from the end of esophagus to the vulva (82.65 ± 4,63 μm) and length of the vagina (173.66 ± 9,81 μm). Additionally, body width in the anus area, in the area of the posterior esophagus (57.09 ± 2,65 μm), and at the anterior end of body (9.43 ± 0.53 μm) can be used to identify female roundworms. We established that the parameters of C. obsignata eggs extracted from feces of the infected chickens are significantly larger than those of eggs obtained from the gonads of female nematodes. Additional indicators (shell thickness, inner and outer egg surfaces) are also proposed, to complement the already existing data. Thus, the nematode eggs isolated from chickens are longer by 18.7% to 25.1% (P < 0.001) and wider by 10.1% (P < 0.01) compared to eggs isolated from gonads. At the same time, the shell thickness of eggs isolated from the chicken feces, and their inner and outer surfaces are also higher by 25.8%, 18.5% and 18.6% (P < 0.001), respectively. Such changes testify to the growth and development of eggs during their migration from the small intestine, where they are excreted by the female, to the external environment along with the avian feces. We obtained new data on the embryogenesis of C. obsignata in laboratory conditions. At 25 °C, the roundworm’s eggs develop to Biosyst. Divers., 25(4) the invasive stage in 12 days with high viability (up to 90.3 ± 1,5%), which contributes to their population growth, rapid cross infection in birds, and survivability of the nematode eggs in the external environment. For the first time, five embryonic developmental stages of C. obsignata are identified, with clear morphological and meristic characteristics, and a stage-by-stage transition from one stage to the next. The first stage, formation of the zygote, morphologically differs by the homogeneous filling of the egg cavity, and is observed in the first two days of cultivation (100.0–88.7%). The second stage, the cleavage of the zygote into morula, is characterized by the formation of unequal blastomeres in the egg cavity during the 4th to 6th days of embryogenesis (66.3–22.0%). The third and fourth stages (bean-like and tadpole embryos) are recognizable by the formation of an embryo shaped like a bean or a tadpole in the egg cavity. These changes were detected mainly on the 6th and 8th days (to 49.6% and 71.6%). The fifth and final stage is characterized by the formation of a mobile larva in the egg cavity, which indicated its viability. On the 10th to 12th days of cultivation, the number of invasive eggs was within 84.3% to 90.3%. Such development of C. obsignata eggs was also confirmed by meristic parameters (length and width, increased by 11.2%, P < 0.01 and 7.5%, P < 0.05, respectively). We added to the already existing data on the embryonic development of C. obsignata, which previously have distinguished only three stages of egg development for this species: eggs without embryo, eggs with a mobile embryo, and invasive larva (Tiersch et al., 2013). Meristic parameters of eggs undergoing embryogenesis are shown for the first time. Conclusion The C. obsignata nematodes, which parasitize chickens, are highly abundant in the central Ukraine (the abundance index was up to 3.4). Female roundworms dominated in the collected material (88.8%). The new species-identifying characters of adult C. obsignata are morphometric. The mature males are identified by the shape, length and width of pseudobursa; distance between its rays; the shape and width of the spicule and spicule sheath in its proximal and distal parts. The females are distinguished by the size of the cuticle vulval lip, which is very variable; the distance between the esophagus end to the vulva; vagina length. The meristic parameters of embryonic stages of C. obsignata are quite variable and depend on the sampled substrate and the stage of development. To facilitate species identification, it is suggested to consider not only the length and width of the eggs, but also their inner and outer surface areas and eggshell thickness. In laboratory conditions, the embryogenesis of C. obsignata occurs in five stages: zygote, morula, bean-like embryo, tadpole embryo, invasive larva. They have significant morphological and meristic specifics. The experimental embryogenesis of C. obsignata occurs in 12 days, and the survival is 90.3%. Acknowlegements The authors are thankful to Dr. Y. I. Kuzmin (Kyiv, Ukraine) for his helpful suggestions. References D’ávila, S., Bessa, E. C. A., & Rodrigues, M. L .A. (2017). Estudo morfométrico e aspectos taxonômicos de Baruscapillaria obsignata (Nematoda, Capillariidae), parasito de Columba livia (Aves, Columbidae). Revista Brasileira de Zoociências, 18(2), 35–44. 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Quantification of Retrograde Axonal Transport in the Rat Optic Nerve by Fluorogold Spectrometry Christian van Oterendorp*, Stavros Sgouris, Michael Bach, Gottfried Martin, Julia Biermann, Jens F. Jordan, Wolf A. Lagrèze University Eye Hospital Freiburg, Freiburg, Germany Abstract Purpose: Disturbed axonal transport is an important pathogenic factor in many neurodegenerative diseases, such as glaucoma, an eye disease characterised by progressive atrophy of the optic nerve. Quantification of retrograde axonal transport in the optic nerve usually requires labour intensive histochemical techniques or expensive equipment for in vivo imaging. Here, we report on a robust alternative method using Fluorogold (FG) as tracer, which is spectrometrically quantified in retinal tissue lysate. Methods: To determine parameters reflecting the relative FG content of a sample FG was dissolved in retinal lysates at different concentrations and spectra were obtained. For validation in vivo FG was injected uni- or bilaterally into the superior colliculus (SC) of Sprague Dawley rats. The retinal lysate was analysed after 3, 5 and 7 days to determine the time course of FG accumulation in the retina (n = 15). In subsequent experiments axona transport was impaired by optic nerve crush (n = 3), laser-induced ocular hypertension (n = 5) or colchicine treatment to the SC (n = 10). Results: Spectrometry at 370 nm excitation revealed two emission peaks at 430 and 610 nm. We devised a formula to calculate the relative FG content (cFG), from the emission spectrum. cFG is proportional to the real FG concentration as it corrects for variations of retinal protein concentration in the lysate. After SC injection, cFG monotonously increases with time (p = 0.002). Optic nerve axonal damage caused a significant decrease of cFG (crush p = 0.029; hypertension p = 0.025; colchicine p = 0.006). Lysates are amenable to subsequent protein analysis. Conclusions: Spectrometrical FG detection in retinal lysates allows for quantitative assessment of retrograde axonal transport using standard laboratory equipment. It is faster than histochemical techniques and may also complement morphological in vivo analyses. Citation: van Oterendorp C, Sgouris S, Bach M, Martin G, Biermann J, et al. (2012) Quantification of Retrograde Axonal Transport in the Rat Optic Nerve by Fluorogold Spectrometry. PLoS ONE 7(6): e38820. doi:10.1371/journal.pone.0038820 Editor: Friedemann Paul, Charité University Medicine Berlin, Germany Received January 13, 2012; Accepted May 11, 2012; Published June 18, 2012 Copyright: ß 2012 van Oterendorp et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: No current external funding sources for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: christian.oterendorp@uniklinik-freiburg.de To assess axonal transport in vivo, tracer substances are introduced into a distinct neuronal area either at the soma or at the axon ending. The amount of tracer that subsequently accumulates at the other end of the neuron is taken as a measure of transport capacity. The detection of the tracer for quantification is most commonly done post mortem on tissue sections using either radioactivity (e.g. I125-BDNF) [5], enzymatic activity (mainly horseradish-peroxidase) [12–14] or fluorescence. [15] A main limitation of this approach is that significant amounts of tracer may get lost during tissue processing. Furthermore, careful sectioning and densitometric analysis are relatively labour intensive and background noise often largely varies between histological sections. To allow a more direct measurement of axonal transport different approaches for in vivo detection of tracers have recently been described. They comprise in vivo retinal imaging of fluorescent tracers (Choe TE et al. IOVS 2011;52: ARVO EAbstract 2448), magnetic resonance imaging with manganese chloride as tracer [11,16,17] or direct observation of fluorescently Introduction A characteristic element of neurons is their axon, which, as in retinal ganglion cells (RGCs), can be several orders of magnitude longer than the cell body. To maintain cellular functions in the remote areas of the axon a transport system is required which carries various cargoes, such as proteins and membrane-encapsulated vesicles from the soma to the axon ending (anterogradely) or in the opposite direction (retrogradely).[1–3] Axonal transport is different from diffusion as the cargo is actively moved by motor protein ATPases which run along a ‘track’ formed by microtubuli or actin. [4]. In the eye, impairment of retrograde axonal transport is considered an important pathogenic factor in glaucoma [5–9], a degenerative disease of retinal ganglion cells (RGCs) which is the second leading cause of irreversible blindness in the world. [10] Besides glaucoma, optic nerve trauma is also characterised by an early breakdown of the axonal transport system. [11]. PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e38820 Axonal Transport Quantification with Fluorogold directed to whole circumference of the aqueous outflow tract located underneath the periphery of the cornea. Laser setting were 100 mm diameter spot size, 500 mW power and 500 ms duration. IOP was measured before and at day 1 and 5 after treatment using a TonoLab device (Thiolat, Finland). tagged probes by in vivo microscopy. [3] However, these methods require considerable investments and highly skilled personnel, which limits their widespread implementation. Here, we report on a robust and technically less demanding method to quantify the retrograde axonal transport capacity in the rat optic nerve by using Fluorogold (FG) as tracer substance, which is spectrometrically detected in retinal lysate. FG is known to be taken up by the cell through endocytosis, presumably pinocytosis, There is no passive diffusion through the cell membrane. Packed in endocytotic vesicles it is actively transported in both directions, ante- and retrogradely. [18] FG has been used for decades as the standard dye for retrograde labelling of RGCs. [15] It shows little fading, is non-toxic in standard concentrations and does not leak from axons while transported. [18]. The method consists of three experimental steps: Superior Colliculus Injection of Fluorogold and Colchicine Animals were deeply anaesthetised with isoflurane. Crystalline Fluorogold (Fluoro-Gold, Fluorochrome, Denver, USA) was resolved as 3% solution in PBS containing 10% DMSO. The skull was exposed and three holes were drilled above the superior colliculus 1 mm lateral to the sagittal and the lambdoidal suture with the central hole being placed at the level of the suture intersection and the other two holes lying 1 mm anterior and posterior to it. A total volume of 3.6 ml FG solution (1.2 ml per hole) was injected using a Hamilton syringe (Hamilton, Bonaduz, Switzerland) mounted on a stereotactic frame (Stoelting, Kiel, Germany). The volume injected into each hole was divided into two equal parts injected at two different levels, 4.2 mm and 4.7 mm below the pia mater. After each injection the needle was left in place for 1 min to avoid reflux of the solution. For disruption of the microtubular network colchicine (or an equal volume of pure PBS for control injections; Sigma-Aldrich, Munich, Germany) was added to the FG solution. The total injection volume for all three holes was 3.9 ml. The solution contained the same amount of FG as for all other injections plus 0.66 ml of a 30 mg/ml colchicine in PBS solution (or 0.66 ml PBS for control injections). 1. Injection of FG into the superior colliculus. 2. Sacrificing of the animal at a specific timepoint, explantation of the retina and lysis of the retinal tissue following standard protocols for western blotting. 3. Spectrometric analysis of the retinal lysate in a fluorescence microplate reader. The resulting emission data provide a direct measure (cFG) for the amount of FG that has accumulated in the retina. Following the spectrometric measurement, the samples can be further used for specific protein analysis e.g. by western blotting. Methods Processing of the Retina Ethics Statement Animals were killed with increasing concentration of CO2 and the eye was excised from the orbit. To explant the retina, the eye was cut open circularly behind the ciliary body to separate cornea and lens from the posterior portion of the eyeball. The retina was carefully detached from the pigment epithelium and fully separated from the sclera by transection of the proximal optic nerve. The retinal tissue was disrupted with an ultrasound probe (Sonopuls, Bandelin; Berlin, Germany) in 150 ml of standard lysis buffer used for western blotting (RIPA buffer containing standard amounts of protease and phosphatase inhibitors (Complete mini, Roche, Germany; plus 200 mM sodium orthovanadate)). The lysate was kept on ice and used immediately for spectrometry. All animals used in this study were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The protocols were approved by the Commission on the Use of Animals in Scientific Procedures of the local government (Tierversuchskommission, Regierungspräsidium Freiburg, Germany; permit number: G-10/106). Animals A total of 33 Sprague Dawley rats, weight 275 to 400 g were used for all in vivo and in vitro experiments. The specifications of the rats used for the in vivo experiments were as follows: Time course of FG accumulation in normal retinae: 13 female rats, weight 300–350 g; optic nerve crush: 3 male rats, weight 275– 300 g, ocular hypertension: 5 male rats, weight 275–300 g, colchicine injection: 10 male rats, weight 275–300 g. Spectrometry of Retinal Lysate For spectrometry the samples, each consisting of 140 ml retinal lysate, were transferred to a black 96 well plate (Greiner Cellstar; Frickenhausen, Germany). All measurements were carried out on an Infinite M200 device (Tecan, Crailsheim, Germany). The excitation wavelength was set to 370 nm. Emission was measured between 400 and 700 nm in 10 nm increments. The gain was set constant throughout all measurements. Optic nerve crush Optic nerve crush was performed as previously described. [19] Briefly, rats were anaesthetised with isofluorane. The orbit was opened through an incision at the superior orbital rim and the optic nerve was approached by partially resecting the lacrimal gland. The optic nerve sheath was cut open longitudinally 1– 2 mm posterior to the globe, while care was taken not to damage blood vessels. The optic nerve was crushed with blunt forceps for 10 s. Before wound closure the retinal perfusion was checked funduscopically. Animals with severe reduction of the perfusion were excluded. Results We have developed a spectrometric approach to reliably quantify FG in retinal tissue lysates and validated the technique in several in vitro experiments. Subsequently, the spectrometric axonal transport measurement was applied to animals with a healthy optic nerve and to animals with experimentally impaired axonal transport for comparison. Laser-induced Ocular Hypertension Model Laser treatment to the trabecular meshwork to rise intraocular pressure (IOP) was performed as previously described. [20] Briefly, after intraperitoneal anaesthesia with Ketamine/Xylazine 60 to 70 laser spots from a frequency doubled 532 nm argon laser were PLoS ONE | www.plosone.org Spectrometric Quantification of FG in Retinal Lysate The spectrum of Fluorogold in retinal lysate exhibits two peaks at 430 and 610 nm. Before studying FG emission, the 2 June 2012 | Volume 7 | Issue 6 | e38820 Axonal Transport Quantification with Fluorogold excitation wavelength providing maximum emission signal was determined using a 1:5000 solution of FG in RIPA lysis buffer. Within a range of 300 to 700 nm excitation wavelength (emission detection at 630 nm) a wavelength of 370 nm showed the highest emission (data not shown) and was therefore used throughout all following experiments. The emission spectrum of pure FG in RIPA lysis buffer exhibits two peaks at 440 and 610 nm (Fig. 1A, dashed line). Pure retinal lysate in standard western blot concentration shows a peak of autofluorescence at 425 nm, which slowly decreases with higher wavelengths (Fig. 1A, black line). Testing RIPA lysis buffer alone revealed very low emission between 400 and 500 nm and no significant emission above 500 nm. (Fig. 1A, grey line). When adding increasing amounts of FG to retinal lysate (Fig. 1B), two peaks emerge at 430 and 610 nm with a trough at 520 nm. (Fig. 1B, white dotted lines) While the peak at 430 nm is a composite result of retinal autofluorescence and FG signal, the second peak at 610 nm is mainly determined by the FG content alone. and the 610 nm peak gradually increases. We hypothesised that variations in the dissected retinal volume would result in an up- or downshift of the emission curve by a certain dilution factor. Hence, normalising the emission curves would eliminate the dilution factor and the slope of the normalised curves should reflect the FG/retinal protein ratio. To test this hypothesis we simulated variations in retinal volume by dilution of retinal lysate containing FG with RIPA lysis buffer and compared the resulting raw and normalised emission spectra. To normalise the spectrum between 520 (trough) and 610 nm (second peak) all emission values (E520 to 610) were divided by the emission value at 520 nm (E520). (Fig. 2 A,B). The raw spectra indicate decreased signal intensities with increasing sample dilution (Fig. 2A). Normalisation of these emission spectra revealed that the slope of the emission curve (E520 to 610) was independent of the sample dilution (Fig. 2B). In contrast, variations of the FG/retinal tissue ratio (Fig. 2C) resulted in different slopes of the normalised emission curve (Fig. 2D). Thus, the curve endpoint (encircled in Fig. 2D), used as a surrogate for the slope, reflects the FG/retinal protein ratio. To quantify the difference in FG content between experimental samples the fact that the normalised emission (en) for lysate containing no FG (en0) is not zero has to be taken into account (see Fig. 2D). The en of a sample containing FG (enx) adds to this baseline value. Thus, to calculate the relative FG content (cFG) of a sample the enx value needs to be normalised to en0. This results in: The relative FG concentration cFG in a sample can be calculated from the peak/trough (610/520 nm) emission. In order to determine the spectrometric parameter that best reflects the FG content in a retinal sample, we conducted in vitro experiments with varying concentrations of retinal protein and FG. When selecting the appropriate parameter from the spectrometric curve, one has to take into account that during excision of the retina from the eyeball, some retinal tissue containing FG is lost, which may vary in different preparations. Consequently, in the analysed sample (which has always a constant volume of sample buffer) the mixture of retinal protein and FG would be more or less diluted. However, given that FG positive RGCs are evenly distributed in the peripheral retina (as demonstrated in supplemental appendix S1 and figure S1) the ratio of FG to retinal protein would always remain constant. For this reason the ideal spectrometric parameter for quantification of the FG content in the retina should reflect the FG/retinal protein ratio rather than the absolute amount of FG in the analysed sample. The 610 nm FG peak emission, however, correlates with the absolute amount of FG and would, therefore, be directly influenced by variations of retinal volume. The spectrometric curves of retinal lysate with different concentrations of FG in Fig. 1B show that with rising FG/retinal protein ratio the slope of the curve between the 520 nm trough cFG ~ with exn ~ exn {e0n e0n x E610 x E520 ðnormalised emission of an FG containing sample xÞ, e0n ~ 0 E610 0 E520 (normalised emission of pure lysate without FG) and Figure 1. Spectrometry of FG and retinal lysate. A) Spectra of pure FG (interrupted line; 1: 5000 dilution) and retinal lysate (black line). The diluent for both was RIPA lysis buffer (grey line). B) Spectra of retinal lysate containing different amounts of FG (white dotted lines). Pure retinal lysate without FG (black dots) and RIPA lysis buffer (grey dots) are shown for comparison. With increasing FG concentration the slope of the curve between 520 and 610 nm increases. doi:10.1371/journal.pone.0038820.g001 PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e38820 Axonal Transport Quantification with Fluorogold Figure 2. Raw and normalised spectra of retinal lysate from untreated animals with FG added in vitro. A and B) Dilution series of retinal lysate containing a constant proportion of FG. 100% is the undiluted sample. 80% and 50% is the content of lysate in the diluted samples. The dilution was done with RIPA lysis buffer. A) Raw emission spectra. The dotted vertical lines mark the 520 to 610 nm range that was normalised in B. B) Spectra after normalisation to the E520 value. The curves of the undiluted and the diluted samples are now congruent, indicating that the normalisation compensates for the effect of different sample dilutions. C and D) Retinal lysate with increasing concentrations of FG. C) Raw emission spectra. The dotted vertical lines mark the 520 to 610 nm range that was normalised in D. D) Spectra after normalisation to the E520 value. The end points of the normalised curves are encircled. They increase with higher FG concentration. doi:10.1371/journal.pone.0038820.g002 Supplemental data (see appendix S2 and figure S2) further characterise the quantitative relation of cFG and the real FG content. cFG relative FG content of the sample: Spectrometric Analysis of FG Retrograde Axonal Transport in the Rat Optic Nerve Consequently, besides the groups of experimental and control retinae which provide the enx data a small group of normal retinae without FG from the same strain, sex and age of rats is necessary in order to obtain the background en0 values. In our experiments the group size of these normal retina samples was n = 3 and 4. The mean of the en0 values was used to calculate the cFG of each experimental sample. Once en0 is determined it can be used for all samples as long as animal strain, sex and age remain unchanged. PLoS ONE | www.plosone.org Time course of FG accumulation in the normal retina. We determined the time course of FG accumulation in the retina by FG injection into the right superior colliculus and measurement of the spectrometric signal in the retinal lysate of the contralateral eye after three, five and seven days. Fig. 3 shows the gradual increase of the normalised curve slope and the cFG value with time. The difference between the control eye group and all three timepoints was statistically significant (p,0.0002; one-way 4 June 2012 | Volume 7 | Issue 6 | e38820 Axonal Transport Quantification with Fluorogold Figure 3. FG accumulation in the retina by retrograde transport over time. Normalised emission curves (A) and relative FG content in the lysate (cFG; B) in control retinal lysate and at three different timepoints after FG injection. The data points and bars are the mean and SEM for each group. The group size is given in panel A. The difference between control and days five and seven are statistically significant (p,0.01 and 0.001, respectively). doi:10.1371/journal.pone.0038820.g003 ANOVA). In post hoc testing at each individual timepoint versus the control eyes, the difference between the control eyes and day five and seven was significant (day three: p.0.05; day five: p,0.01; day seven: p,0.001; Bonferroni’s multiple comparison test). As the difference between control and day three was not statistically different in the post hoc analysis we chose day five as the timepoint appearing most appropriate to detect differences in FG transport in the following in vivo experiments. p = 0.006). In the optic nerve crush group cFG was reduced to levels of FG-free controls (Fig. 4 A,B). In the ocular hypertension model with a less severe optic nerve damage, a smaller reduction in FG levels was detected. In the colchicine group, a moderate but highly significant drop in axonal transport capacity was detected (Fig. 4). After spectrometric measurement the retinal lysate can be further used for western blotting. The FG itself as well as the spectrometry procedure do not seem to cause any interference with the subsequent western blot (Fig. 4G). When probing for phospho-Erk1/2 there was no indication for increased protein degradation or dephosphorylation. Spectrometric quantification of axonal transport impairment. To assess whether the method is capable of detecting axonal transport impairment, we conducted three independent in vivo experiments. Optic nerve damage was induced by either: 1. optic nerve crush for an acute severe axonal injury, 2. ocular hypertension for a more mild axonal damage or 3. colchicine injection in the superior colliculus for pharmacological disruption of the microtubuli network in the optic nerve axons. For optic nerve crush (n = 3) or laser treatment to induce ocular hypertension (n = 5) FG was injected into both superior colliculi and the damaging treatment was performed unilaterally during the same anaesthesia. In the laser treated eyes the intraocular pressure at day one was 34.865.3 mmHg (mean and SEM) higher than in the contralateral control eye. As known for this laser model, the pressure had returned to almost normal level at day five (1.263.3 mmHg above control). For microtubular disruption, colchicine (or PBS for control) was injected together with FG into the right superior colliculus (n = 5 for treatment and control group). The spectrometric measurements were done at the left eye as the contralateral eye is predominantly affected in albino animals due to crossing of more than 90% of the axons. In all cases the cFG value was determined from retinal lysate five days after treatment. The en0 used for all calculations was obtained by averaging the measurements of four retinae without FG (en0 = 0.32460.014). All three modes of damage showed significantly less FG in the retinal lysates of treated eyes compared to untreated control eyes (Fig. 4 A-F; cFG mean 6 SEM for untreated and treated eye; one-tailed t-test: optic nerve crush: 1.8560.5 and 20.00560.004, p = 0.029; ocular hypertension: 1.8060.19 and 1.5360.22, p = 0.025; colchicine: 1.8060.06 and 1.3060.10, PLoS ONE | www.plosone.org Discussion We have developed a method to quantify the retrograde axonal transport capacity in the rat optic nerve by fluorogold (FG) superior colliculus injection and subsequent spectrometric detection of FG in retinal lysate. The relative FG concentration in the tissue lysate, cFG, is calculated from the spectrometric emission measured at 610 and 520 nm in the experimental sample and a FG-free reference. A significant increase of FG signal above baseline was detected from day five after superior colliculus injection. Therefore, this timepoint was chosen in transport interruption studies. Choosing day seven might have resulted in a slightly stronger signal with better differentiation of different grades of transport impairment. However, we found choosing the earliest significant timepoint more sensible because the timecourse of FG accumulation shows a steep rise from day three to five and levels off after day five. This suggests saturation of the neurons and increasing anterograde transport of FG from the soma back to the axon ending at later time points. Also, the FG concentration at the superior colliculus slowly decreases with time as a consequence of diffusion into larger brain areas and drainage to the blood vessels. Therefore, choosing the most dynamic phase of FG accumulation in the retina provides the highest chance to detect a transport impairment in neurodegeneration. For rapidly developing neurodegeneration, even the five day observation time may be too long. But for more slowly progressing disease models like chronic glaucoma or generalised 5 June 2012 | Volume 7 | Issue 6 | e38820 Axonal Transport Quantification with Fluorogold Figure 4. FG accumulation in the retina after different types of optic nerve injury. A-F) Normalised emission curves (A, C, E) and cFG (B, D, F) five days after optic nerve injury. Data points and error bars are mean and SEM. A, B) Optic nerve crush, n = 3, p = 0.029. C, D) Laser induced ocular hypertension, n = 5, p = 0.025. E, F) Colchicine injection to the superior colliculus, n = 5 for treatment and control group, p = 0.006). G) Western blot for phospho-Erk1/2 from control retinal lysate after superior colliculus injection of PBS (lane ‘‘C’’) and from retinal lysate used for FG spectrometry (lane ‘‘F’’). Both lanes show a similar band pattern indicating that despite the FG labelling and spectrometric measurements the retinal lysate can be further used for western blots. doi:10.1371/journal.pone.0038820.g004 different timepoints each followed by spectrometry five days later would result in a value that is not only dependent on the number of physically present RGCs (like RGC counts) but would also reflect the RGC’s ability to carry out axonal transport. This approach, however, requires careful validation which was beyond the scope of the present work. Besides the much shorter data acquisition time another advantage of FG spectrometry over histological detection methods is that the tissue lysates can be used for further analyses. The FG present (molecular weight approx. 500 Daltons) does not interfere with SDS-Gel electrophoresis and wet-blotting procedures as demonstrated in Fig. 4G. Given that axonal transport impairment is considered a key pathogenic factor in a variety of neurodegenerative diseases such as amyotrophic lateral sclerosis, [21] X-linked Charcot-Marie Tooth neuropathy [22] and hereditary spastic paraplegias [23] FG spectrometry could be applied to any nerve and any direction of transport. In cases where cargo binding is affected, such as in Parkinson’s and Alzheimer’s disease [24], different cargo vesicles may be differentially affected. Thus, the degree of transport impairment of FG carrying vesicles may be different from pathogenically relevant vesicles, for example those carrying neurotrophic factors. [25] In all other cases where the disease primarily affects the movement of the motor proteins (dynein or kinesin, e.g. Huntigton’s disease) or the tubular ‘railroad’ (microtubuli) we consider a similar impact on the different types of endocytotic vesicles. If both FG injection area and target area are located in the brain they need to be anatomically well separated to avoid tracer CNS diseases it appears adequate. Even if some RGCs die and release their FG during the observation period, the tracer would still appear in the lysate and be available for detection. By normalisation of the raw spectra our method compensates for variations in the retinal volume retrieved (Fig. 2). This eliminates the need for a separate method to acquire the sample protein concentration. The normalised emission curve of diluted samples was similar to undiluted samples up to a dilution of 50% (Fig. 2 A,B). This would control for a much higher variability of protein concentration than observed in typical experiments. In a study measuring the weight of nine consecutive retinae dissected in our laboratory we determined a variation coefficient of 11%. A source of error when using the spectrometric method would be the comparison of groups injected with different preparations of FG working dilutions. Variations in pipetting as well as fading of fluorescence over time may lead to erroneous results. Therefore, each experimental group of animals needs an individual control group injected with the same FG solution, preferably at the same day. The primary goal in developing the method was to measure axonal transport as a functional parameter. It was not designed to replace RGC counts for neurodegeneration or neuroprotection studies. Although the number of RGCs and the amount of FG in the retina somehow correlate, spectrometry does not distinguish between FG in viable RGCs and FG released from dead RGCs. However, by carefully choosing the timepoint of FG injection and sampling, the spectrometric measurement may provide additional valuable information about RGC survival. For example, in a slowly progressing neurodegenerative disease FG injections at PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e38820 Axonal Transport Quantification with Fluorogold spillover by interstitial diffusion. The fact that cFG corrects for variations in tissue protein concentration is particularly helpful when working with neuronal projections to the brain, as tissue volumes dissected from a particular brain area often vary largely. However, this does not exclude sampling error. If a significant amount of the dissected tissue is outside the neuron projection zone the cFG will be falsely low. In contrast to optical in vivo imaging systems which are increasingly used in eye research, FG spectrometry uses ex vivo tissue (retinal lysate) which requires a higher number of animals when investigating different timepoints. Information on spatial differences in the retinal transport between individual RGCs or areas of the retina are lost. However, using a highly sensitive, wavelength-specific and well calibrated detection system, which by its measurement principle excludes a large number of variables occurring in optical imaging in vivo, the spectrometric method is particularly suitable for quantification purposes. This is also reflected by the very low inter-measurement variability of the in vitro measurements shown in shown in supplemental figure S2. Our data suggest, that FG spectrometry may serve as a valuable addition to in vivo imaging. Furthermore, FG spectrometry provides a robust and low-cost tool to study axonal transport in the optic nerve as a stand-alone system if more elaborate and expensive techniques are not available. retina and the counting frame (dashed line), which is divided into a central and peripheral sub-frame. The size of the counting frame was enlarged for better visibility. B) Box plot of the peripheral to central subframe RGC density (p/c ratio) of 4 retinae. The dashed line indicates a p/c ratio of 1 (no difference between central and peripheral sub-frame). There is no statistically significant difference to the value 1 (p = 0.56) indicating that RGC density does not change as a function of eccentricity in the outer periphery. C) Representative image of FG labelled RGCs. Image size equals counting frame size. The horizontal white line indicates the boundary of the sub-frames. Top peripheral part, bottom central part. (TIF) Figure S2 Relationship between relative and absolute FG content. cFG plotted against rFG of retinal lysate with known amounts of FG added. The data points are the mean and SEM of n = 3 samples. The interrupted line is the linear regression of the data points. The plot demonstrates that the experimentally determined cFG value is proportional to the real FG concentration rFG. (TIF) Acknowledgments Supporting Information The authors are grateful to Günther Schlunck for critically reading of the manuscript and Ms. Sylvia Zeitler for her excellent technical assistance. Appendix S1 Assessment of RGC density changes in the outer periphery of the retina. (DOC) Author Contributions Conceived and designed the experiments: CvO GM JFJ WAL. Performed the experiments: CvO SS JB. Analyzed the data: CvO MB GM JB. Contributed reagents/materials/analysis tools: MB WAL. Wrote the paper: CvO. Appendix S2 Calculation of the relative FG content. (DOC) Figure S1 Quantification of RGC density changes in the outer periphery of the retina. A) Schematic of a flatmounted References 14. Kuypers HG, Kievit J, Groen-Klevant AC (1974) Retrograde axonal transport of horseradish peroxidase in rats forebrain. Brain Res 67: 211–218. 15. Catapano LA, Magavi SS, Macklis JD (2008) Neuroanatomical tracing of neuronal projections with Fluoro-Gold. Methods Mol Biol 438: 353–359. 16. Chan KC, Fu QL, Hui ES, So KF, Wu EX (2008) Evaluation of the retina and optic nerve in a rat model of chronic glaucoma using in vivo manganeseenhanced magnetic resonance imaging. Neuroimage 40: 1166–1174. 17. Matsuda K, Wang HX, Suo C, McCombe D, Horne MK, et al. (2010) Retrograde axonal tracing using manganese enhanced magnetic resonance imaging. Neuroimage 50: 366–374. 18. Schmued LC, Fallon JH (1986) Fluoro-Gold: a new fluorescent retrograde axonal tracer with numerous unique properties. Brain Res 377: 147–154. 19. Grieshaber P, Lagreze WA, Noack C, Boehringer D, Biermann J (2010) Staining of fluorogold-prelabeled retinal ganglion cells with calcein-AM: A new method for assessing cell vitality. J Neurosci Methods 192: 233–239. 20. van Oterendorp C, Lorber B, Jovanovic Z, Yeo G, Lagreze WA, et al. (2011) The expression of dynein light chain DYNLL1 (LC8–1) is persistently downregulated in glaucomatous rat retinal ganglion cells. Exp Eye Res 92: 138–146. 21. Bilsland LG, Sahai E, Kelly G, Golding M, Greensmith L, et al. (2010) Deficits in axonal transport precede ALS symptoms in vivo. Proc Natl Acad Sci U S A 107: 20523–20528. 22. Vavlitou N, Sargiannidou I, Markoullis K, Kyriacou K, Scherer SS, et al. (2010) Axonal pathology precedes demyelination in a mouse model of X-linked demyelinating/type I Charcot-Marie Tooth neuropathy. J Neuropathol Exp Neurol 69: 945–958. 23. Reid E, Kloos M, Ashley-Koch A, Hughes L, Bevan S, et al. (2002) A kinesin heavy chain (KIF5A) mutation in hereditary spastic paraplegia (SPG10). Am J Hum Genet 71: 1189–1194. 24. Morfini GA, Burns M, Binder LI, Kanaan NM, LaPointe N, et al. (2009) Axonal transport defects in neurodegenerative diseases. J Neurosci 29: 12776–12786. 25. Mufson EJ, Kroin JS, Sobreviela T, Burke MA, Kordower JH, et al. (1994) Intrastriatal infusions of brain-derived neurotrophic factor: retrograde transport and colocalization with dopamine containing substantia nigra neurons in rat. Exp Neurol 129: 15–26. 1. Hirokawa N, Niwa S, Tanaka Y (2010) Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease. Neuron 68: 610–638. 2. Levy JR, Holzbaur EL (2006) Cytoplasmic dynein/dynactin function and dysfunction in motor neurons. Int J Dev Neurosci 24: 103–111. 3. Salinas S, Bilsland LG, Schiavo G (2008) Molecular landmarks along the axonal route: axonal transport in health and disease. Curr Opin Cell Biol 20: 445–453. 4. Kapitein LC, Hoogenraad CC (2010) Which way to go? Cytoskeletal organization and polarized transport in neurons. Mol Cell Neurosci 46: 9–20. 5. Pease ME, McKinnon SJ, Quigley HA, Kerrigan-Baumrind LA, Zack DJ (2000) Obstructed axonal transport of BDNF and its receptor TrkB in experimental glaucoma. Invest Ophthalmol Vis Sci 41: 764–774. 6. Vrabec JP, Levin LA (2007) The neurobiology of cell death in glaucoma. Eye (Lond) 21 Suppl 1: S11–14. 7. Johnson EC, Guo Y, Cepurna WO, Morrison JC (2009) Neurotrophin roles in retinal ganglion cell survival: lessons from rat glaucoma models. Exp Eye Res 88: 808–815. 8. Quigley HA (1995) Ganglion cell death in glaucoma: pathology recapitulates ontogeny. Aust N Z J Ophthalmol 23: 85–91. 9. Chidlow G, Ebneter A, Wood JP, Casson RJ (2011) The optic nerve head is the site of axonal transport disruption, axonal cytoskeleton damage and putative axonal regeneration failure in a rat model of glaucoma. Acta Neuropathol 121: 737–751. 10. Quigley HA, Broman AT (2006) The number of people with glaucoma worldwide in 2010 and 2020. Br J Ophthalmol 90: 262–267. 11. Haenold R, Herrmann KH, Schmidt S, Reichenbach JR, Schmidt KF, et al. (2011) Magnetic resonance imaging of the mouse visual pathway for in vivo studies of degeneration and regeneration in the CNS. Neuroimage 59: 363–376. 12. Johansson JO (1988) Inhibition and recovery of retrograde axoplasmic transport in rat optic nerve during and after elevated IOP in vivo. Exp Eye Res 46: 223– 227. 13. Lynch G, Gall C, Mensah P, Cotman CW (1974) Horseradish peroxidase histochemistry: a new method for tracing efferent projections in the central nervous system. Brain Res 65: 373–380. PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e38820 

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ARCHIVES OF ACOUSTICS Vol. 45, No. 1, pp. 103–110 (2020) DOI: 10.24425/aoa.2020.132486 Research Paper Side Effect of the Use of Acoustic Barriers Observed in the Infra Range Andrzej STANIEK, Cezary BARTMAŃSKI∗ Central Mining Institute Department of Acoustics, Electronics and IT Solutions Pl. Gwarków 1, 40-166 Katowice, Poland e-mail: astaniek@gig.eu ∗ Corresponding Author e-mail: cbartmanski@gig.eu (received April 1, 2019; accepted October 5, 2019) Acoustic barriers which are positioned along traffic lanes are designed to protect the surroundings from excessive noise. Such structures are to reverberate, diffract and damp the propagating acoustic waves. However, this method of shielding has some disadvantages which include constraint visibility and structure-born noise. The interaction between traffic-caused movement of air mass and acoustic barriers may generate infra noise waves. That is undesirable and should be estimated. The authors undertook the research to diagnose the plausible side effect of structure-born noise of such barriers because it may influence human body (Kasprzak, 2014). As a mechanical structure, the acoustic barrier is characterized by mechanical parameters which, in the field of modal analysis, are made up of natural frequencies, damping factors and mode shapes. In this paper the authors investigated the acoustic pressure distribution in the neighborhood of a real acoustic barrier in the scope of infra noise propagation. The methods of modal analysis were used to identify natural frequencies of the barrier and dominating frequencies of propagating waves in the far field. The correlation between observed vibration and acoustic signals is presented. Keywords: modal analysis; infra range noise; vibration; acoustic barrier; road traffic. 1. Introduction 2. Field of interest Methods of measurement and analysis may be divided into two types. One group is connected with direct acoustic field measurements and the other with structure mechanical parameters identification. Both have advantages and disadvantages. Accordingly, accessibility of the object under test is quite different. In the following paper two different methods of structural behaviour analysis are compared and correlation of results is discussed. One of the methods uses infra range acoustic microphones positioned in different points with one taken as the reference. The correlation of signals measured in different points and their power spectrums were calculated. The other is a classical modal analysis method (Remington, 1997), where measurement of vibrating structure – acoustic barrier was performed with the use of a laser scanning vibrometer. As the source of vibration both an impact hammer and road traffic environmental influence were utilized. The aim of the research was to evaluate the magnitude of acoustic waves in the infra range and identify the source of generation of that signal near acoustic barriers. It is important to establish whether the propagating waves get to a point positioned behind a noise barrier as a secondary effect of vibrating surface of the shielding construction or they are generated by other sources positioned in the neighborhood of measurement points. There are many areas where researchers investigate the influence of infra range waves on human body, e.g. wind turbines (Carlile et al., 2018). Also, reduction of continuous-cycle stationary low-frequency tone-like noise propagated through diffraction over a barrier border is limited (Borchi et al., 2016). Accordingly, different methods to measure and analyze these phenomena have been incorporated (Degan, 2003; Serraris, 2016). The problem of reducing noise in low frequency range is well known and difficult to 104 Archives of Acoustics – Volume 45, Number 1, 2020 solve (Peiró-Torresa et al., 2016). In this paper a comparison between vibration response of acoustic barriers and acoustic field measurements using low frequency range microphones is presented. These methods utilize modal analysis procedures and are used in order to realize the targeted study. 3. Methods Three methods were used to measure the plausible effect of reverberated noise emission of acoustic barriers. The first approach was based on measurements of vibration of the acoustic barrier, mainly the shielding surface, utilizing laser scanning vibrometer. The investigated acoustic barrier and distribution of measurement points are presented in Figs 1 and 2. marked as 2, attached using an adapter plate, and near the ground – marked as 3, cemented to the bottom part of the fence. In all positions the vibrations were measured in the horizontal direction, perpendicular to the acoustic barrier. The third measurement approach was a pure acoustic one with low frequency microphones localized at a distance of 8 m behind the acoustic barrier under test. The measurement points are shown in Fig. 2. There was one reference microphone localized at point 5 (at height of 1.6 m), and three rowing microphones positioned sequentially at points 4, 6, 7, 8, and 9 (at height of 1.6 m and 3.9 m). Additionally two points were localized close to the acoustic barrier at height 1.6 m and 3.9 m, positioned in the gap space of the investigated structure, not shown in Fig. 2. The scanning points are marked as 1, the investigated acoustic barrier as 2 and the laser scanning vibrometer as 3. The measurements were performed using Polytec PSV-400 laser scanning vibrometer and Brüel & Kjær PULSE measuring system. In measurement chain PCB accelerometers type 393B12 and GRAS microphones type 40AN, and 40AZ were used. The results of these approaches were analyzed and compared. 4. Results Fig. 1. The investigated noise barrier and distribution of measurement points. To describe a mechanical structure behaviour completely the following models may be used: a spatial model – represented by mass, stiffness and damping matrixes, a response model – consisting of all the frequency response functions for all degrees of freedom, and a modal model – covering all the mode shapes and natural frequencies. These models are equivalent to each other. In experimentally – based investigations it is almost impossible to match their accuracy. So we use reduced models, which means that the complete structural model of the investigated object of N × N matrix is related to n × n model of response matrix and m × n model of modal matrix, where N ≫ n > m. General rules for relevance and adequacy of complete and incomplete models for the response, modal and spatial models are as follows (Ewins, 2000): Fig. 2. The investigated acoustic barrier and distribution of measurement points. The second approach was based on both vibration and acoustic measurements, i.e. signals from two microphones positioned at the far field at a distance of 8 m behind the acoustic barrier were correlated with vibration signal from the reference accelerometer positioned at different places, shown in Fig. 1, on the shield – marked as 1, attached using glue mass, on the pillar – where letter N represents full dimension of analyzed model of the structure, n and m refer to the number A. Staniek, C. Bartmański – Side Effect of the Use of Acoustic Barriers Observed in the Infra Range of measured response functions and modal vectors respectively. Moreover, as all the results were obtained using experimental modal analysis, in cases where both microphones and accelerometers were used, the reciprocity theorem condition was not fulfilled. Although it is one of the modal analysis basic assumptions, we must be aware that in such a situation it cannot be achieved for practical reasons. However, the aim of this study was not to give full description of mechanical behaviour and dynamic properties of the investigated structure as well as to describe its spatial model. The investigation 105 was performed to show the correlation between different approaches to vibroacoustic measurements of the tested object and seemed to be adequate for scientific analysis. The main purpose of investigations was to diagnose the propagating noise behind the acoustic barrier and to answer the question: what the source of that noise is, especially in the infra range and whether it is the effect of response of acoustic barriers to mechanical excitation. In Tables 1 to 9 the results of analysis performed for the aforementioned situations are presented. Table 1. Comparison of the results of scanning method of two repeated measurements, the reference piezoelectric accelerometer attached to a pillar using stud and sticking mass. Frequency 1 Frequency 2 Difference Damping 1 Damping 2 Difference 9.77 10.20 0.43 0.43 0.53 0.10 12.12 11.95 0.17 0.65 3.29 2.64 16.19 15.95 0.24 0.04 1.75 1.71 16.71 15.95 0.76 0.05 1.75 1.70 27.90 – – 0.20 – – 33.28 33.28 0.00 0.04 0.03 0.01 Table 2. Comparison of the results of scanning method, a change of the reference piezoelectric accelerometer placement – on the pillar versus in the ground. Frequency 3 Frequency 4 Difference Damping 3 Damping 4 Difference 12.12 12.02 0.10 0.65 1.81 1.16 16.71 16.56 0.15 0.05 0.66 0.61 27.90 28.33 0.43 0.20 0.34 0.14 33.28 – – 0.04 – – Table 3. Comparison of the results of a scanning method using laser vibrometer with piezoelectric accelerometer positioned on the pillar as a reference versus the results of measurement performed at the same time using 2 microphones positioned in the far field (approximately 8 m from the acoustic barrier) with the reference accelerometer positioned on the screen. Frequency 5 Frequency 6 Difference Damping 5 Damping 6 Difference 9.77 8.94 0.83 0.43 0.91 0.48 12.12 11.88 0.24 0.65 0.59 0.06 24.62 24.91 0.31 0.08 0.59 0.51 33.28 33.31 0.03 0.04 0.13 0.09 Table 4. Comparison of the results of a scanning method using laser vibrometer with piezoelectric accelerometer positioned on the pillar as a reference versus the results of measurement performed at the same time using 2 microphones positioned in the far field (approximately 8 m from the acoustic barrier) with the reference accelerometer positioned on the pillar. Frequency 7 Frequency 8 Difference Damping 7 Damping 8 Difference 9.77 10.36 0.59 0.43 0.09 0.34 12.12 12.05 0.07 0.65 0.08 0.57 16.71 16.22 0.49 0.05 0.10 0.05 24.62 – – 0.08 – – 33.28 33.29 0.01 0.04 0.09 0.05 106 Archives of Acoustics – Volume 45, Number 1, 2020 Table 5. Comparison of the results of a scanning method using laser vibrometer with piezoelectric accelerometer positioned on the pillar as a reference versus the results of measurement performed at the same time using 2 microphones positioned in the far field (approximately 8 m from the acoustic barrier) with the reference accelerometer positioned in the ground. Frequency 9 Frequency 10 Difference Damping 9 Damping 10 Difference 9.77 9.24 0.53 0.43 1.06 0.63 12.12 11.46 0.66 0.65 0.17 0.48 16.71 – – 0.05 – – 23.45 23.39 0.06 0.24 0.18 0.06 27.90 27.02 0.88 0.20 0.10 0.10 33.28 32.88 0.40 0.04 0.06 0.02 Table 6. Comparison of the results of a scanning method using laser vibrometer versus the results of measurement performed at the same time using 2 microphones positioned in the far field (approximately 8 m from the acoustic barrier), in both cases the reference accelerometer positioned in the ground. Frequency 11 Frequency 12 Difference Damping 11 Damping 12 Difference 8.93 9.24 0.31 0.77 1.06 0.29 12.02 11.46 0.56 1.81 0.17 1.64 12.30 11.46 0.84 0.32 0.17 0.15 16.56 – – 0.66 – – 17.17 18.15 0.98 0.47 0.06 0.41 28.33 – – 0.34 – – Table 7. Comparison of the results of measurement with microphones positioned at 6 points in the far field (approximately 8 m from the acoustic barrier, one of microphones used as a reference) versus the results of measurement performed using 2 microphones positioned in the far field (approximately 8 m from the acoustic barrier) with the reference accelerometer attached to the screen. Frequency 13 Frequency 14 Difference Damping 13 Damping 14 Difference 6.59 – – 1.82 – – 12.51 11.88 0.63 0.25 0.59 0.34 14.32 14.18 0.14 0.86 0.48 0.38 18.81 – – 0.18 – – 30.09 30.10 0.01 0.16 0.11 0.05 33.36 33.31 0.05 0.10 0.13 0.03 34.63 – – 0.24 – – Table 8. Comparison of the results of measurement with microphones positioned at 6 points in the far field (approximately 8 m from the acoustic barrier, one of microphones used as a reference) versus the results of measurement performed using 2 microphones positioned in the far field with the reference accelerometer mounted on the pillar. Frequency 15 Frequency 16 Difference Damping 15 Damping 16 Difference 6.59 – – 1.82 – – 12.51 12.05 0.46 0.25 0.55 0.30 14.32 13.49 0.83 0.86 0.55 0.31 18.81 18.70 0.11 0.18 0.19 0.01 30.09 30.10 0.01 0.16 0.11 0.05 33.36 33.29 0.07 0.10 0.09 0.01 34.63 – – 0.24 – – A. Staniek, C. Bartmański – Side Effect of the Use of Acoustic Barriers Observed in the Infra Range 107 Table 9. Comparison of the results of measurement with microphones positioned at 6 points in the far field (approximately 8 m from the acoustic barrier, one of microphones used as a reference) versus the results of measurement performed using 2 microphones positioned in the far field with the reference accelerometer mounted in the ground. Frequency 17 Frequency 18 Difference Damping 17 Damping 18 Difference 6.59 – 1.82 – – 12.51 12.85 0.34 0.25 0.41 0.16 14.32 13.70 0.62 0.86 0.37 0.49 18.81 18.15 0.66 0.18 0.06 0.12 30.09 – – 0.16 – – 33.36 33.28 0.08 0.10 0.13 0.03 34.63 – – 0.24 – – We may group the following tables as comparison between: • measurements of microphone signals with reference to one microphone signal in the far field. • laser scanning measurements with reference point attached to different parts of the acoustic barrier, • measurements of microphone signals with reference to a vibration signal measured at points placed on the acoustic barrier, Additionally, to analyze results of those approaches more precisely, statistics tools were used and correlation as well as linear regression were calculated, as shown in Figs 3 to 7. Fig. 3. Correlation and linear regression, Tables 1 and 2. Fig. 4. Correlation and linear regression, Tables 3 and 4. 108 Archives of Acoustics – Volume 45, Number 1, 2020 Fig. 5. Correlation and linear regression, Tables 5 and 6. Fig. 6. Correlation and linear regression, Tables 7 and 8. Fig. 7. Correlation and linear regression, Table 9. In order to check the correlation between acoustic response (in the far field) and vibration excitation of the investigated acoustic barrier, the measurements and consequently modal analysis were performed using 2 microphones positioned in the far field (approximately 8 m from the acoustic barrier) with the reference accelerometer positioned in sequence on the screen, on the pillar and in the ground, shown in Fig. 1. That is not a typical use of modal analysis (see the comment above regarding the use of both microphones and accelerometers). These measurement results were compared with results taken from the scanning method – the comparison between the first and second type of measurements, aforementioned in the text. The results of comparison are presented in Tables 3–6 and in Figs 4 and 5. Looking for relationships between the above results we have observed that in almost all cases, Tables 1 to 9, except Table 6, there are diagnosed modes of frequencies of about 12 Hz and 33 Hz (matched with green shadow colour). It implies that these modes are characteristic of both the analyzed acoustic barrier A. Staniek, C. Bartmański – Side Effect of the Use of Acoustic Barriers Observed in the Infra Range as well as of propagating acoustic waves, and consequently that these modes are excited by the acoustic barrier itself. For a frequency of about 16 Hz (marked out in blue shadow colour) we may imply that it is the mode characteristic of the pillar vibration, not observed in the remaining cases. The frequencies of about 14 Hz, 18 Hz and 30 Hz (marked out in orange shadow colour) seem to be generated by other sources positioned in the neighboring environment. Other modes may have more local character. The diagnosed frequencies are generally well correlated. In two cases the correlation is worse: Tables 6 and 9, where the comparison is made with measurement performed with 2 microphones positioned in the far field with the reference accelerometer mounted near the ground. That might be explained by the fact that there is a poorer correlation between reference accelerometer and microphone signals. It should also be mentioned that in some cases all frequencies could not have been detected so they are missing from particular tables. This might result from not exciting or measuring the corresponding frequencies. Considering that the results were obtained with the usage of different approaches of exciting the investigated structure to vibration as well as with different response and reference signals, we may infer that for frequencies of about 12 Hz and 33 Hz it is the inner feature of an acoustic barrier that causes such an effect, i.e. generates these frequencies in the infra range. The measured levels at low frequency range using microphones exceeded 70 dB and the recorded noise was generated mainly by heavy and long vehicles. Figure 8 shows the waterfall diagram of one of the laser scan where the vibration of acoustic barrier under test was measured. Figure 9 presents the laser scanning vibrometer head and distribution of points of performed scan. 109 Fig. 9. Laser scanning vibrometer and investigated noise barrier. Distribution of measurement points. 5. Conclusions Taking into account the above results, we may imply that acoustic barriers generate acoustic waves in the infra range as a response to traffic transport, which is an undesirable side effect of reducing noise in a protected area. Though it is difficult to assess the impact of low frequency noise on human body, it influences citizens living in the neighboring area. The research made by Central Institute for Labour Protection (Kaczmarska et al., 2008) revealed that infrasonic noise at workplaces in offices requiring employee’s special attention focus cannot exceed 86 dB for 8 hours duration. Here the influence of analyzed phenomena is permanent and exceeding 70 dB, but not all diagnosed modes may be attributed to acoustic barriers as a source of infra range noise. Nevertheless, it is suggested the constructors should consider designing such protecting shields with resonant frequencies well above the infra range (Ishizuka, Fujiwara, 2003; Borchi et al., 2016). Also the analysis for other types of acoustic barriers should be performed. References 1. Borchi F., Carfagni M., Martelli L., Turchi A., Argenti F. (2016), Design and experimental tests of active control barriers for low-frequency stationary noise reduction in urban outdoor environment, Applied Acoustics, 114: 125–135, doi: 10.1016/ j.apacoust.2016.07.020. 2. Carlile S., Davy J.L., Hillman D., Burgemeister K. (2018), A review of the possible perceptual and physiological effects of wind turbine noise, Trends in Hearing, 22: 1–10, doi: 10.1177/2331216518789551. Fig. 8. Cross spectrum functions for operational modal analysis of the object under test. 3. Degan A. (2003), Acoustic barriers: performance and experimental measurements, Tecniacustica, Bilba. 110 Archives of Acoustics – Volume 45, Number 1, 2020 4. Ewins D.J. (2000), Modal Testing: theory, practice and application, Research Studies Press Ltd., Letchworth, Herts, England. 5. Ishizuka T., Fujiwara K. (2003), Performance of noise barriers with various edge shapes and acoustical conditions, Kyushu Institute of Design. 6. Kaczmarska A., Augustyńska D., Łuczak A. (2008), Infrasonic noise at workplaces in offices requiring employee’s special attention focus [in Polish: Hałas infradźwiękowy na stanowiskach pracy wymagających koncentracji uwagi – propozycja kryterium uciążliwości], Occupational Safety. Science and Practice, 2008(7–8): 28–32, http://archiwum.ciop.pl/28638. 7. Kasprzak C. (2014), The influence of infrasounds on the Sensory Motor Rhythm (SMR) of EEG Sig- nal, Forum Acusticum, 7–12 September 2014, Cracow, Poland. 8. Peiró-Torres M.P., Redondo J., Bravo J.M., Sánchez Pérez J.V. (2016), Open noise barriers based on sonic crystals. Advances in noise control in transport infrastructures, Transportation Research Procedia, 18: 392–398, doi: 10.1016/j.trpro. 2016.12.051. 9. Remington P.J. (1997), Experimental and theoretical studies of vibrating systems, Encyclopedia of Acoustics, Vol. 2, John Wiley & Sons. 10. Serraris J. (2016), Propagation of sound around different noise barrier models using a three-dimensional scan based sound visualization technique, Noise Control Engineering Journal, 64(2): 134–141, doi: 10.3397/ 1/376366. 

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Psychological Distress and Social Support among Conflict Refugees in Urban, Semi-rural and Rural Settlements in Uganda: Burden and Mechanisms of Association Stephen Lawoko (  s.lawoko@gu.ac.ug ) Gulu University Faculty of Medicine https://orcid.org/0000-0003-4369-7955 Catherine Nakidde Centre for Health and Socio-economic Improvement Eric Lugada Centre for Health and Socio-economic Improvement Maria Ssematiko Centre for Health and Socio-economic Improvement Dunstan P. Ddamulira Agency for Cooperation and Research in Development (ACORD) Andrew Masaba Lutheran World Federation Brian Luswata Directorate of Governance and Regulation, inistry of Health and Community Service: Ministry of Health Community Development Gender Elderly and Children Eric A. Ochen Makerere University College of Humanities and Social Sciences Betty Okot Makerere University College of Humanities and Social Sciences Denis Muhangi Makerere University College of Humanities and Social Sciences Gloria Seruwagi Makerere University College of Humanities and Social Sciences Research Keywords: Psychological Distress, Social Support, MHPSS, COVID-19, Urban/Rural, Conflict Refugees, Uganda Posted Date: June 16th, 2021 Page 1/26 DOI: https://doi.org/10.21203/rs.3.rs-606878/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 2/26 Abstract Background: Recent research shows that psychological distress is on the rise globally as a result of the COVID-19 pandemic and restrictions imposed on populations to manage it. We estimated the burden of psychological distress and scrutinized the role that social support may play in moderating psychological distress among conflict refugees in urban, semi-rural and rural settlements in Uganda during the COVID19 pandemic. Methods: Cross-sectional survey data on psychological distress, social support, demographics, socioeconomic and behavioral variables was gathered from 1014 adult refugees randomly sampled from urban, semi-rural and rural refugee settlementsin Uganda, using two-staged cluster sampling. Research instruments were checked for reliability using Cronbach’s Alpha. T-test, Analysis of Variance (ANOVA) and Multivariable Linear Regression were applied to analyse the data in SPSS-version 22. Statistical significance was assumed at p<0.05. Results: Refugees resident in rural/semi-rural settlements exhibited higher levels of psychological distress, lower levels of social interaction and lower levels of social attachment than peers resident in urban settlements. However, they reported, on the other hand, higher adequacy of social interaction and higher adequacy of social attachment than peers resident in urban settlements. Adequacy of social interaction and attachment were directly associated with psychological distress and acted as stress buffers in the relationship between psychological distress and rural/urban settlement. Conclusions: There is a settlement-inequality (i.e. rural vs. urban) in psychological distress and social support among conflict refugees in Uganda. Both the direct and buffering effects of adequacy of social interaction and attachment can explain the rural/urban discrepancy in psychological distress, with important implications for Mental Health and Psychosocial Support (MHPSS) interventions. Introduction COVID-19 has exasperated the health and wellbeing of population groups worldwide since it was declared a pandemic over a year ago. Beside the fear, anxiety, confusion, and frustrations triggered by the pandemic per se [1], the stringent measures activated by governments to curtail the disease spread have further exacerbated psychological distress. Globally, experts predict that the collective impact of these restrictions on household socio-economics, health and wellbeing, as well as the social infrastructure of communities will extend beyond the lifespan of the pandemic [1–7]. These trends are further manifest in the increasing burden of intimate partner violence, depression and anxiety [8–12]. Despite the growing body of evidence suggesting deteriorating psychosocial wellbeing at population level, equivocal data from refugee populations is lacking. Currently surpassing 80 million in number globally, conflict refugees are particularly at heightened risk of psychological distress when contrasted with host communities. A myriad of psychosocial problems including Post-Traumatic Stress Disorder (PTSD), depression and anxiety [13–14], and psychological distress triggered by fears of apprehension, Page 3/26 risk of deportation, difficult living conditions, poor access to health, social, communication, financial and legal services [15–21], have been reported in refugee settings. This is in addition to the stressors imposed by restrictions to contain COVID-19 pandemic at the population level [1–7]. An assessment of psychological distress status and psychosocial support needs for refugees is therefore particularly imperative in the pandemic era. Social support can play an important role in preventing, containing or moderating the psychological wellbeing of populations through several mechanisms. While some scholars have emphasized its role as a stress buffer [22–23], others have highlighted its protective function as a coping facilitator [24–25] as well as its role in health promotion [26]. As a stress buffer, social support is envisioned to alleviate the detrimental impact of stressful life events by modifying negative appraisals and promoting problem solving strategies [24, 26–27]. Some scholars argue however that during times of severe or chronic distress, the buffering effect of support may be limited [28–29]. In contrast, as a coping facilitator, social support is envisaged to provide regular directly rewarding experiences such as positive affection, which prevent the development of psychologically distressful outcomes [22, 26]. Moreover, a supportive network is hypothesized to promote behaviors beneficial to health such as timely seeking of healthcare and adaptation of healthy lifestyles (e.g. healthy nutritional choices and physical activity) [24, 26–27], thereby reducing the likelihood of psychological distress. Although population studies have alluded to a breakdown in social support networks during the COVID-19 pandemic, there is a dearth of studies investigating its role in containing psychological distress particularly in refugee populations where the need is augmented. Moreover, refugees live under varying conditions with some residing in urban and others in rural settings, and this has implications on vulnerability to health problems. For instance, it could be assumed that urban refugees may be more prone to social support by virtue of the vast availability of social networks or opportunities for social interaction (e.g. through gainful employment) on the one hand, but may on the other hand be confronted with challenges related with psychological stressors that accompany increased population density and diversity in cities such as unemployment, violence, marginalization and exposure to health-risk behaviours [30–32]. Moreover, settlement of refugees in rural/urban areas have in some countries been characterised by cultural homogeneity, and culture is known to play an important role in social integration. In Uganda the context of the current study for example, South Sudanese, Congolese and Somali refugees have been resettled in rural, semi-rural and urban settlements respectively. While on the one hand the collectivist orientation of such settlements may promote social interaction by virtue of shared cultural norms and interest, efforts to integrate into a new society or culture may discourage such intentions [33–34]. Thus, an assessment of the nature of relationship between social support and psychological distress in distinct settlements (i.e. urban vs. rural) is warranted on its own right. Upon this background, we scrutinized social support and psychological distress, and compared the impact of social support on psychological distress among refugees in urban, semi-rural and rural settlements in Uganda. Specifically, we addressed the following research questions: Page 4/26 1. How do refugees in urban, semi-rural and rural settlements in Uganda rate their levels of psychological distress during the COVID-19 pandemic? In other words, what is the burden of psychological distress among refugees in urban, semi-rural and rural settlements in Uganda? 2. How do refugees in urban, semi-rural and rural settlements in Uganda rate their availability and adequacy of social support during the COVID-19 pandemic? 3. What is the mechanism linking social support to psychological distress? Does social support impact directly (independently) on psychological distress and/or Does social support buffer psychological distress resulting from urban/rural residency among refugees? Methods Study Context Uganda is host to nearly1.5 million conflict refugees from neighbouring countries of Rwanda, Burundi, South Sudan and Democratic Republic [35]. These refugees exhibitpoor living conditions, pre-existing mental and psychosocial challenges [36–40]. The inaction caused byCOVID-19 preventive measures, starting from the first case identification on 21 March 2020, including restrictions on mass gatherings, public transport, entry and exit at border points, and lockdown on several social services [41] is envisioned to have impacted further on psychological distress in this already vulnerable population. Indeed, emerging data in vulnerable groups of refugee women and slum-dwellers have catapulted their risk to stigma, all forms of violence and financial disadvantage [42–43].While researchers have predicted the economic, psychosocial, physical, and other consequences of COVID-19 on refugees/migrants in Uganda and beyond based on previous epidemics [44–47], there is a dearth of evidence on the burden of psychological distress, social support and mechanisms linking these phenomena in refugee settings during the COVID-19 pandemic. The current work intends to fill this gap in the evidence using Uganda as a case study. Such data could be useful in the design of interventions to cushion psychosocial problems among refugees through the modification of social support agents. Study site and population We conducted the research at 3 large refugee settlements in different regions of Uganda, hosting over 400,000 refugees: 1. Kisenyi, refugee settlement, an urban refugee setting in the centre of the capital city (Kampala) hosting over 70,000 refugees of mainly Somali origin. The refugees live integrated with their host. 2. Kyaka II refugee Settlement in the South Western part of Uganda, a semi-rural refugee setting hosting multinational refugees from the Democratic Republic of Congo (DRC), Burundi and Rwanda totaling approximately 124,000 refugees. The refugees live partly segregated from their host but with freedom of movement and shared services. The region can be considered as semi-rural, with a blend of rural and urban activities (e.g. farming, industrial activities) 3. Adjumani refugee settlement in North-West Nile Uganda, hosting about 214,000 refugees predominantly of South Sudanese nationality. The refugees live rather segregated from their host but Page 5/26 with freedom of movement and shared services. The region is considered as rural, with Agriculture as main activity. Study design Cross-sectional survey data on various health and social indicators was gathered from 1014 refugees randomly selected from each of the study sites. For the current study, data on psychological distress, social support, demographic, social and behavioral indicators was of primary interest. Sampling procedure Participants were sampled using a two-staged cluster sampling procedure in each settlement. The first stage involved selecting clusters of zones in the main settlement using systematic random sampling with probability proportional to zone size (PPS). The second stage constituted systematic sampling of households in selected zones. Random numbers procedures were used to choose one adult household member (i.e. 15 years and above) from among all adults in the household to constitute the final participant. This procedure resulted in 1014 refugees, with the following distribution among the settlements: Adjumani n = 342;Kyaka 354; Kisenyi n = 318. Ethical Considerations Thirty Research Assistants (RAs) were trained to collect data using mobile tablets, in a bid to reduce interindividual contact and the risk of COVID-19 spread during interviews. The training oriented RAs on the purpose of the study; ethical considerations; data collection methods and tools; COVID-19 prevention, symptoms, measures and precautions; and standard operating procedures (SOPs) in fieldwork in light of COVID-19. The training also involved testing of the data collection tool among a purposively selected refugee sample of n = 30 in each of the 3 settlements, from zones neighboring but not included in the main study. Slight adjustments were made to data collection tools following this exercise. Informed consent was received from all participants and confidentiality considered. The potential risk and benefits of the study were explained to all participants and in light of the heightened risk of COVID-19 transmission, we developed Standard Operational Procedures (SOPs) for protection of refugees as well as data collectors, guided by Safety and Security Strategy for COVID-19 of the World Health Organization (WHO) and Uganda Ministry of Health COVID-19 Guidelines. The study was approved by the Makerere University School of Public Health Institutional Review Board (MakSPH IRB) and the Uganda National Council of Science and Technology (UNCST), the two bodies governing academic research in Uganda. Additionally, the Ministry of Health (MoH), Kampala Capital City Authority (KCCA) and the Office of the Prime Minister, which is in charge of refugee affairs, gave clearance for execution of the study. Data collection tools and Study variables Page 6/26 A comprehensive questionnaire covering several areas of relevance to public health and COVID-19 was developed. For the current study, the following variables were of interest: Dependent variables The dependent variable for the study was psychological distress, measured using Kessler’s Psychological Distress Scale (K10)[48], a previously validated 10-item instrument measuring distress in terms of feelings of nervousness, hopelessness, tiredness, restlessness, fidgety, depressed mood, sadness, worthlessness, cheerlessness and loss of effort, during the past 14 days, with a 5-level response ranging from none of the time (score 1) to all of the time (score 5). A composite score for depression is calculated for each participant as the sum of the 10 items. Thus, individual scores for depression scale ranged from 10–50, with higher scores indicative of higher depression risk. Cronbach’s alpha testing for internal consistency/reliability of Kessler’s Psychological Distress Scale for the current sample was 0.91 indicating very high reliability. Independent variables The main independent variable for this study was social support, with the aim to assess its direct impact on psychological distress, together with the assessment of its role in buffering the impact of living in various settlement categories (i.e. urban, rural, semi-rural settlement) on distress. Social support was measured using the Interview Schedule for Social Integration (ISSI) [49], which assesses social support in terms of the Availability and Adequacy of Social Interaction and Social Attachment. Availability of Social Interaction was assessed using six items inquiring of participants to indicate the number of people: with whom they have common interest, meet and talk to regularly, can speak with openly, can borrow things from and can turn to when in trouble. This was thus a continuous scale ranging between 0-infinity. The participants were in addition requested to rate the Adequacy of these numbers indicating whether they desire more (coded as 1), less (coded as 1) or no change (coded as zero) in the number indicated. Thus, Adequacy of Social Interaction was rated on a total scale ranging between 0–6, with higher scores denoting higher inadequacy (i.e. poor adequacy). Cronbach’s alpha testing for internal consistency/reliability of Availability and Adequacy of Social Interaction respectively for the current sample was 0.50 and 0.81 respectively, indicating moderate to high reliability respectively. Availability of Social Attachment was assessed based on six items inquiring of participants to indicate using a “Yes” (coded as 1) or “No” (coded as 0) response regarding whether there is someone special: from whom they derive support, they feel close to, they share happy moments, they can embrace for comfort, appreciates what they do, and with whom they can share inner thoughts. For social attachment, composite individual scores are calculated as the sum of responses to each item. Thus, scores for social attachment range between 0–6, with higher scores representing higher availability. Adequacy of Social Attachment was assessed by inquiries to participants on whether they desire more (coded 1), less (coded 1) or no change (coded 0) regarding the special persons mentioned above. Thus, scores for this scale ranged between 0–6, with higher scores representing higher inadequacy (i.e. poor adequacy). Cronbach’s Page 7/26 alpha testing for internal consistency/reliability of Availability and Adequacy of social attachment respectively for the current sample was 0.55 and 0.87 respectively, indicating moderate to high reliability respectively. Other independent variables included in the study were demographic, social and behavioral characteristics of participants, such as sex, age, marital status, religion, income, employment status, alcohol and substance use and physical activity. As these variables are from previous studies generally known to be associated both with social support and psychological distress, it is prudent to adjust for them in the main analyses in a bid to control for possible confounding. Statistical Analysis Cronbach’s Alpha coefficients were calculated to assess for reliability of the instruments of interest (i.e. Kessler’s Psychological Distress Scale and ISSI)in the current sample. In the assessment and contrasts of the burden of psychological distress in rural, semi-rural and urban refugee populations (research question 1), ANOVA was used. To compare the rates of availability and adequacy of social support among refugees in rural, semi-rural and urban settlements (research question 2), Analysis of Variance (ANOVA) were used. To assess for bivariate associations between Psychological distress and Social support indicators, demographic, social and behavioural independent variables, t-test, ANOVA and Pearsons Correlations tests were run. In the assessment of the mechanism linking social support to distress (i.e. the direct effects and the stress buffering effects, research question 3), Multivariable Linear Regression (MLR) was used. In the assessment of the direct effect of Social Support on Psychological Distress, MLR including the entire study sample was run with psychological distress as the dependent variable and social support indicators as the main independent variables, but adjusting for independent effects of demographic (including urban/semi-rural/rural residency), social and behavioural factors on the dependent variable. The direct effect of social support would be confirmed if the social support indicators remained statistically significantly associated with distress despite adjustment for possible confounders. The stress buffering effect was assessed using stratified MLR with settlement as the main independent variable, but controlling for demographic, social and behavioural factors. Stratification was by social support availability/adequacy levels categorized under “high” (defined as lacking in 1 or none of the 6 dimensions studied), “moderate”(i.e. lacking in 2 or 3 of the 6 dimensions studied), “low” (lacking in 4 of the 6 dimensions studied) and “very low” (lacking in 5 or 6 of the 6 dimensions studied). Social support would be considered as a stress buffer if it acted as an effect modifier in the association between psychological distress and urban/semi-rural/rural residency, i.e. if the association between refugee settlement (i.e. rural/urban residency) and psychological distress is stronger where support is weak and weaker where support is strong. SPSS version 22 was used for all analyses and a statistical significance of p < 0.05 was assumed for all tests. Page 8/26 Results Demographic, social and behavioural characteristics of Participants Study participants were equally distributed across the rural, semi-rural and urban settlements of Adjumani, Kyaka IIand Kisenyi respectively (table 1). Majority of participants were of South-Sudanese, Congolese or Somali origin, with a specific nationality predominating in each settlement (i.e. the urban settlement of Kisenyi was dominated by Somalis (100%), rural settlement of Adjumani dominated by South Sudanese (99%), and semi rural settlement of Kyaka II dominated by Congolese (97%).). Majority of the refugees were: aged under 45 years (over 80%), of female sex (65%), Protestants (36%) and Moslems (30%). Many refugees were unemployed (70%), in the low-income bracket (i.e. 57% earning less than 50,000 Uganda Shillings per week) and were uneducated (40%). Concerning behavioral characteristics, few participants were smokers (4%) or drank alcohol (7%), but many (44%) were physically inactive by way of exercise. Participants ratings of Availability and Adequacy of Social Interaction by Settlement On average, refugees rated their availability of social interaction rather high, ranging between 23-28 people across the settlements (table 2), with significantly higher availability ratings observed among refugees in urban followed by rural and lowest in semi-rural settlements respectively [f (2, 1011)=6.05; p<0.005]. On the other hand, refugees rated adequacy of social interaction rather low (i.e. 3.9-4.2), on a scale of range 0-6. As higher scores denote higher grade of inadequacy, significantly higher adequacy of social interaction was observed for semi-rural residents followed by rural and lowest among urban residents respectively [f (2, 1011) = 54.1; p<0.001]. In summary, availability of social interaction was highest among refugees in urban settlements when contrasted with peers in rural and semi-rural settlements. On the other hand, adequacy of social interaction was higher among semi-rural and rural residents than urban peers. Page 9/26 Refugees ratings of Availability and Adequacy of Social Attachment Overall and by Settlement On average, refugees rated their availability of social attachment rather high (on average ranging between 5.2 – 5.4) on a scale of range 0 – 6) (table 2), with significantly higher ratings observed among urban followed by rural and lowest among semi-rural residents respectively [f (2, 1011) = 47.9; p<0.001]. On the other hand, refugees rated adequacy of social attachment rather low (i.e. 4.1-4.3). As higher scores denote higher grade of inadequacy, significantly higher adequacy of social attachment was observed for semi-rural residents followed by urban and lowest among rural residents respectively [f (2, 1011) = 50.5; p<0.001]. Overall, availability of social attachment was higher among refugees in urban settlements when contrasted with peers in rural and semi-rural settlements. On the other hand, adequacy of social interaction was higher among semi-rural and urban residents than rural peers. Refugees ratings of Psychological Distress Overall and by Settlement Levels of distress were on average moderate, ranging between 20.4-21.5 across settlements on a scale ranging from 10 - 50 (table 2). As higher scores denote higher distress, significantly higher psychological distress was observed among semi-rural followed by rural and lowest among urban residents respectively [f (2, 1011) = 47.9; p<0.001]. Association between Psychological Distress and demographic, social and behavioural factors Psychological distress levels varied significantly according to demographic, social and behavioural characteristics of refugees (table 3). Refugees of Congolese nationality, followed by Rwandese, Burundians and South Sudanese respectively exhibited significantly higher distress than refugees of Somali origin [f (3, 1010) = 35.3; p<0.001] (table 3).These results mirror the previously presented findings (table 2) indicating that psychological distress varied between settlements, with higher distress traced to Semi-rural (where Page 10/26 Congolese, Rwandese and Burundians are predominant) followed by rural (where SouthSudanese are predominant) and lowest in Urban (where Somalis are predominant) settlements respectively. Female refugees reported, on average, higher distress levels than male peers [t (1010) = 1.95; p = 0.05]while Protestants, Catholics and other religions exhibited on average higher distress levels than Moslems [f (3, 1010) = 28.3; p<0.001]. Unemployed refugees exhibited significantly higher psychological distress levels than peers in employment activity or studying [f (4, 1009) = 7.0; p<0.001]. Pearsons Correlation Coefficients (r) showed that psychological distress increased with increasing age [r=0.18; p<0.01], reduced with increasing level of education [r= -0.20; p<0.001], and reduced with increasing level of physical activity [r= -0.25; p<0.001] among the refugees. Bivariate association between Social Interaction, Social Attachment and Psychological Distress There was significant correlation between Psychological distress levels and social support indicators (table 3). Adequacy of social interaction exhibited a negative correlation with psychological distress (i.e. distress levels reduced as adequacy of social interaction increased) [r= -0.18; p<0.01]. Availability of social attachment exhibited a negative correlation with distress (i.e. psychological distress levels reduced as availability of social attachment increased) [r= -0.18; p<0.01]. Similarly, as adequacy of social attachment increased, distress levels reduced [r= -0.16; p<0.01]. Multiple Linear Regressions Analysis assessing the direct effect of Social Support on Psychological Distress As indicated in Table 4, Adequacy of Social Interaction and Adequacy of Social Attachment were associated with Psychological Distress after controlling for plausible demographic, social and behavioural confounders. With increasing inadequacy in both respects, Psychological Distress increased. Availability of Social Interaction and Availability of Social Page 11/26 attachment on the other hand were not significantly associated with Psychological Distress after controlling for plausible demographic, social and behavioural confounders. Thus, the direct effect of Social Support on Psychological Distress hypothesis was confirmed for Adequacy but not for Availability of support networks. Stratified Multiple Linear Regressions Analysis assessing the buffering effect of Social Support in the association between Settlement and Psychological Distress Since Availability of Social Interaction and Availability of Social Attachment were not independently associated with Psychological Distress in the previous regressions analyses, they are not included in the stratified analyses. In the following analyses therefore, stratified analyses are run for Adequacy of Social Interaction and Adequacy of Social Attachment respectively. As indicated in Table 5, the association between settlement and psychological distress remained statistically significant in the overall and in stratified analyses according to levels of Adequacy of Social Interaction. However, the strength of association particularly for the rural/urban contrasts increased progressively between the stratified analyses for high (i.e. B=0.21), moderate (i.e. B=0.30), and low (i.e. B=0.32) levels of Adequacy of Social Interaction (expressed by the size of the standardized regression coefficients B). A similar trend was observed with regard to the analyses stratified by levels of Adequacy of Social Attachment, i.e. the strength of association particularly for the rural/urban contrasts increased progressively between the stratified analyses for high (i.e. B=0.09), moderate (i.e. B=0.25), and low (i.e. B=0.29) levels of Adequacy of Social Attachment. These findings are congruent with the stress buffering hypothesis i.e. the association between settlement (i.e. rural/urban residency) is stronger where adequacy of social interaction and adequacy of social attachment respectively is low and weaker where adequacy of such support is high. Page 12/26 Discussion Few studies in refugee settings have scrutinised the individual level social resources available to refugees and how these may manifest on their psychological wellbeing. Accordingly, we assessed the burden of psychological distress and scrutinized the mechanisms linking social support to distress among refugees resident in urban, semi-rural and rural settlements in Uganda. Burden of Psychological Distress in Urban, Semi-rural and Rural Refugee Settlements Levels of psychological distress were on average moderate among refugees, but with notable variations across settlements, i.e. significantly higher distress levels were observed among refugees resident in semi-rural and rural settings when contrasted with peers resident in urban settlements. While such data, to the best of our knowledge is previously lacking in refugee cohorts, population studies have generated contradictory results in this respect. Some scholars have supported the notion of heightened psychological distress in urban areas due to stressors related with urbanization such as unemployment, violence, marginalization, discrimination and increased exposure to health-risk behaviours, while other researchers have envisioned residents in urban settings to have an upper edge in health as a result of better access to health and social services in urban than in rural settings [30–32]. Our findings could be a reflection of the latter circumstances. Availability and Adequacy of Social Interaction and Social Attachment in Urban, Semi-rural and Rural Refugee Settlements Refugees rated their availability of social interaction (which encompassed access to resources required for regular social activities such as conversation and meeting people) and availability of social attachment (which covered access to social relations of emotional relevance such as embracing for comfort and sharing inner thoughts) as high, but rated adequacy of such resources (i.e. whether such resources were perceived sufficient) as low. Moreover, the results from the regressions analyses found adequacy of social interaction/attachment rather than availability of social interaction/attachment to correlate positively with psychological distress (i.e. with increasing adequacy, levels of distress reduced). Corroborating previous research in general population studies [50–51], these findings contribute new data to the refugee literature demonstrating that the effectiveness of social support in moderation of psychological distress among refugees is anchored on the perceived meaning and value of the support network (here measured by adequacy of social interaction and attachment), rather than the support network’s morphological aspects (e.g. density and size of the network). Availability of social interaction and attachment was highest among refugees residing in urban settlements when contrasted with peers residing in rural and semi-rural settlements. A plausible explanation for this result could stem from the fact that urban refugees lived in integrated rather than separate settlements with the host communities, and this may have provided opportunities for social networking with host community members, to a higher extent than in rural settlements where the residential areas are distinctively for refugees. On the other hand, adequacy of social interaction and Page 13/26 attachment was highest among refugees resident in semi-rural and rural settlements when contrasted with peers resident in urban settlements. These results are more difficult to reconcile. While the findings linking availability of support networks to urban settlement are largely congruent with previous works in general populations [30–32], the findings linking adequacy of support to rural settings appear to contradict those works. Social support is a multi-dimensional concept and discrepancies in results across studies could stem from variations in its conceptualization between the studies. For instance, some studies have measured social support in terms of the form of support received (e.g. emotional support, instrumental support, cognitive guidance, informative and appraisal support) [52–53], while others have focussed on the morphology of the network (e.g. size) [50–51], or value of relationships within the network (e.g. relationship reciprocity) [54]. Thus, cross-study comparisons should be done with caution. Mechanisms Linking Social Support to Psychological Distress in Urban, Semi-rural and Rural Refugee Settlements The regressions analyses ruled out availability of social interaction and availability of social attachment respectively as independent predictors of Psychological Distress, but confirmed adequacy of social interactions and adequacy of social attachment respectively as predictors of such distress, i.e. Psychological Distress levels reduced as adequacy of social interaction and adequacy of social attachment respectively increased. Furthermore, our study generated evidence in support of the stress buffering function of social support [22–23], which postulates that the deleterious effects of psychosocial stressors on health can be reduced or eliminated with the existence of a strong support network. Specifically, we hypothesized and demonstrated that social support can moderate the impact of stressors due to rural/urban settlement on psychological distress in refugee populations. Implications and Recommendations The burden of psychological distress and social support in refugee settings drum for the need of surveillance of these phenomena as a basis for informed action. The need to revitalize, strengthen or restructure existing social networks cannot be overemphasised, with observance of COVID-19 prevention measures notwithstanding. Humanitarian actors will need to invest more in mental health and psychosocial support services (MHPSS), tailoring these interventions to the unique needs of different refugee categories. The leveraging of professionals across disciplines (e.g. social workers, psychologists, community health-workers) to be more actively involved in the MHPSS response to distress is apparent, particularly in rural/semi-rural refugee settings, during this COVID-19 era where refugees’ access to the natural individual support networks (e.g. relatives and friends) have been temporarily constrained by government directives. A multi-sectoral team including main actors in humanitarian settings, should be set up to address how such measures can be implemented without necessarily exposing refugees to increased risk of COVID-19 alongside other challenges to implementation. Moreover, the findings confirming the direct and buffering hypotheses of social support have important implications for interventions to manage distress in refugee settings. Firstly, the direct pathway linking specific aspects of social support (i.e. inadequacy) to psychological distress provides new evidence to Page 14/26 support the design of focused (as opposed to generic) interventions, aimed at strengthening social support among refugees. In addition, the study has demonstrated the potential utility of the stress buffer framework of social support in the identification of irreversible stressor in refugees’ lives (e.g. settlement in urban/rural settings) that could lead to psychological distress. Moreover, social support could, within such frameworks, be applied to predict the thresholds at which such stressors may lead to severe psychological distress, information of importance in early detection and prevention strategies. Study Limitations While the study carried the strength of a large representative sample of refugees in Uganda, utilized validated instrumentation and applied conventional statistical methodology to respond to the research questions, some of its weaknesses deserve acknowledgement. First, the cross-sectional design does not permit inference of causality, which is implied in some of the hypotheses tested. For instances, we can only firmly ascertain associations between social support and distress. Secondly, the homogenous cultural composition of refugees in each of the studied settlements (i.e. predominance of Somalis in the urban settlement vs. South Sudanese and Congolese in the rural and semi-rural settlements respectively) makes it difficult to disentangle associations between distress and urban/rural settlement on the one hand from associations between distress and country of origin on the other hand. Thus, the observed differences in distress between settlements could be masking differences in distress due to nationality. Due to the presence of collinearity in our data with regard to these two variables (i.e. settlement and nationality), we could not address this question by way of analysis. Future research focussing on the separation of these two effects is warranted using the appropriate study design. Conclusion There is a settlement inequality (i.e. rural vs. urban) in psychological distress and social support among conflict refugees in Uganda. Both the direct and the buffering effects of adequacy of social interaction and attachment can be forwarded to explain the rural/urban discrepancy in psychological distress, with important implications for Mental Health and Psychosocial Support (MHPSS) interventions. Declarations Ethics approval and consent to participate The study was approved by the Makerere University School of Public Health Institutional Review Board (MakSPH IRB) and the Uganda National Council of Science and Technology (UNCST), the two bodies governing academic research in Uganda. Informed consent was obtained from each participant. Consent for publication Not Applicable Page 15/26 Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests None to declare Funding The study was funded through a research grant received by the authors from Research for Health in Humanitarian Crises (R2HC)/Elrha, supported bythe UK Foreign, Commonwealth and Development Office (FCDO),Wellcome, and the National Institutefor Health Research (NIHR). Authors' contributions SL and GS conceptualized this study. EL, MS, DD, AM, BL, EO, BO and DM were responsible for data collection, cleaning and management at respective refugee settlements. SL and CL performed the literature review. SL and EL performed the statistical analyses. SL, CL and GS wrote the first draft of the paper. All authors contributed to the consequent drafts draft of the paper. Acknowledgements The authors are grateful to R2HC/Elrha and funding partners for financial support tothe project “REFugee Lived Experiences, Compliance and Thinking (REFLECT) in COVID-19”, of which the current study is part. References 1. Dubey S, Biswas P, Ghosh R, Chatterjee S, Dubey MJ, Chatterjee S, Lahiri D, Lavie CJ. Psychosocial impact of COVID-19. Diabetes metabolic syndrome. 2020;14(5):779–88. https://doi.org/10.1016/j.dsx.2020.05.035. 2. Hamza Shuja K, Aqeel M, Jaffar A, Ahmed A. COVID-19 pandemic and impending global mental health implications. PsychiatriaDanubina. 2020;32(1):32–5. 3. Josephson A, Kilic T, Michler JD. (2021). 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Figures. at a glance https://www.unhcr.org/figures-at-a-glance.html (accessed May 2021). 38. Ahimbisibwe F. Uganda and the refugee problem: Challenges and opportunities. Institute of Development Policy and Management (IOB), University of Antwerp, Working Paper No. 2018 May. 39. Monteith W, Lwasa S. The participation of urban displaced populations in (in) formal markets: contrasting experiences in Kampala, Uganda. Environment Urbanization. 2017;29(2):383–402. Page 18/26 40. Peltzer K. Trauma and mental health problems of Sudanese refugees in Uganda. The Central African journal of medicine. 1999 May 1;45(5):110-4. 41. Hovil L. Self-settled refugees in Uganda: An alternative approach to displacement? Journal of Refugee Studies. 2007 Dec;20(4)(1):599–620. 42. Kaiser T. Between a camp and a hard place: rights, livelihood and experiences of the local settlement system for long-term refugees in Uganda. Journal of Modern African Studies. 2006 Dec 1:597–621. 43. Ministry of Health. (2020). COVID-19 response info hub. Available from: https://covid19.gou.go.ug/timeline.html. 44. Nakalembe JS. (2020). Covid 19 interventions verses a woman in Uganda [Internet]. Stop Gender Based Violence [cited 2021 Apr 20]. Available at:https://www.cehurd.org/covid-19-interventionsverses-a-woman-in-uganda/. 45. Gato J. (2020). The Effects of COVID-19 Lockdown on Urban Refugees in Kampala. [Internet]. Available at: https://blog.fluchtforschung.net/covid-19-lockdown-on-urban-refugees-in-kampala/. 46. Bukuluki P, Mwenyango H, Katongole SP, Sidhva D, Palattiyil G. The socio-economic and psychosocial impact of Covid-19 pandemic on urban refugees in Uganda. Social Sciences Humanities Open. 2020;2(1):100045. 47. Guadagno L. Migrants and the COVID-19 pandemic: An initial analysis. Geneva: International Organization for Migration; 2020. p. 60. 48. Truelove S, Abrahim O, Altare C, Lauer SA, Grantz KH, Azman AS, Spiegel P. The potential impact of COVID-19 in refugee camps in Bangladesh and beyond: a modeling study. PLoS Med. 2020;17(6):e1003144. 49. Tsamakis, K., Tsiptsios, D., Ouranidis, A., Mueller, C., Schizas, D., Terniotis, C.,… Rizos, E. (2021). COVID19 and its consequences on mental health. Experimental and therapeutic medicine, 21(3), 1– 1. 50. Kessler RC, Andrews G, Colpe LJ, Hiripi E, D K MR, T NORMAND SL, WALTERS EE, Zaslavsky AM. Short screening scales to monitor population prevalences and trends in non-specific psychological distress. Psychological medicine. 2002 Aug 1;32(6):959. 51. Henderson S, Duncan-Jones P, Byrne DG, Scott R. Measuring social relationships the interview schedule for social interaction. Psychological medicine. 1980 Nov;10(4):723–34. 52. Kaplan BH, Cassel JC, Gore S. Social support and health. Medical care. 1977 May 1;15(5):47–58. 53. Berkman LF, Syme SL. Social networks, host resistance, and mortality: a nine-year follow-up study of Alameda County residents. 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Tables Table 1: Demographic, Social and Behavioural Characteristics of Participants Page 20/26 Characteristic %1 n Refugee Settlement Adjumani (Rural) 342 33.8 354 35.0 317 31.2 South Sudanese (99% in Rural) 343 33.9 Congolese (97% in Semi-rural) 342 33.8 308 29.8 Kyaka II (Semi-rural) Kisenyi (Urban) Nationality Somali (100% in Urban) Others(Rwandese, Burundians) (80% in Semi-rural) 26 2.5 Age 15-24 220 21.7 25-34 355 35.0 35-44 254 25.1 45-54 84 8.3 55-64 67 6.6 65-74 25 2.5 75-84 8 0.8 Sex Male 318 31.4 Female 693 68.4 307 30.3 188 18.6 370 36.5 Religion Moslem Catholic Protestant Other (e.g. Adventist, Jehovah Witness) 14314.6 Occupation Employed 34 3.4 Page 21/26 Self-employed 120 11.9 Unemployed 712 70.3 113 11.2 Student Other (shifting e.g. vendor, retailer etc) 32 3.1 Less than 50,000 577 57.1 50,000 – 100,000 87 8.7 35 3.5 33 3.3 407 40.2 Primary level 303 29.9 Secondary level 230 22.7 24 2.4 46 4.5 Yes 38 3.8 No 970 95.8 Yes 72 7.2 No 931 91.9 Earnings per week 100,000 - 200,000 Over 200,000 Highest Education Level No Education Tertiary or vocational University Smoker Drink Alcohol Exercise Never 1 450 44.4 Once 175 17.3 2-3 times 258 4 times or more 127 24.5 12.5 Percentages may not add up to 100% due to missing values Table 2: Refugees ratings of levels of Social Support and Psychological Distress: Overall and by settlement Page 22/26 Variable Rural Semi-rural Urban Total Mean (CI) Mean (CI) Mean (CI) Mean (CI) 25.6 (21.7-29.5) 20.8 (17.2-24.4) 30.9 (26.5-35.5) 25.9 (23.6-28.3) 4.3 (4.1-4.5) 3.1 (2.9-3.4) 4.6 (4.4-4.8) 4.0 (3.9-4.2) 5.3 (5.0-5.5) 4.9 (4.8-5.1) 5.7 (5.5-5.9) 5.3 (5.2-5.4) 4.7 (4.5-4.8) 3.2 (2.9-3.5) 4.6 (4.4-4.8) 4.2 (4.1-4.3) 21.2 (20.5-22.4) 23.7 (22.7-24.8) 17.3 (16.5-18.0) 20.9 (20.4-21.5) (Range) Availability of Social Interaction** (0 – infinity) Adequacy of Social Interaction*** (0 – 6) Availability of Social Attachment*** (0 – 6) Adequacy of Social Attachment*** (0 - 6) Psychological Distress*** (10 – 50) **ANOVA f-test statistically significant at p<0.01; *** ANOVA f-test statistically significant at p<0.001 Table 3: Bivariate association between demographic, socio-behavioural and social support indicators and Psychological Distress Page 23/26 Characteristic Mean (SE) Nationality*** South Sudanese 21.4 (0.5) Congolese 23.7 (0.5) Somali 16.9 (0.3) Other (i.e. Rwandese, Burundians) 23.6 (1.8) Sex* Female Male 21.3 (0.3) 20.1 (0.5) Religion*** Moslem Catholic Protestant Other (e.g. Adventist, Jehovah Witness) 16.9 (0.4) 22.4 (0.7) 23.3 (0.5) 21.1 (0.7) Occupation*** FormalEmployed 17.5 (1.3) Self-employed 18.7 (0.6) Unemployed 21.9 (0.3) Student Other (shifting e.g. vendor, retailer etc) 18.7 (0.8) 19.8 (1.3) Pearsons Correlation Coefficient (r) Age (Years) ** 0.18 Earnings per week (Shillings) -0.02 Highest Education Level** -0.20 Smoking (Yes/No) -0.04 Page 24/26 Drink Alcohol (Yes/No) -0.02 Exercise (Frequency)** -0.25 Availability of Social Interaction -0.04 Adequacy of Social Interaction** -0.18 Availability of Social Attachment** -0.18 Adequacy of Social Attachment* -0.06 *T-test for difference in means or Correlation Coefficient (r) statistically significant at p<0.05; ** ANOVA f-test or Correlation Coefficient (r) statistically significant at p<0.01 Table 4: Adjusted Standardized Regression Coefficients (Standardized Beta) assessing independent associations between Social Support indicators and Psychological Distress Characteristic Standardized Beta1 1 Availability of Social Interaction -0.02 Adequacy of Social Interaction** 0.11 Availability of Social Attachment -0.02 Adequacy of Social Attachment** 0.10 The analysis was adjusted for settlement, sex, religion, occupation, age, education level and physical activity. Due to high collinearity between nationality and settlement, nationality was excluded from the analysis Table 5: Adjusted Standardized Regression Coefficients (B) assessing buffering effect of Social Support indicators in the association between Settlement and Psychological Distress Page 25/26 Un-stratified Analysis Analysis Stratified by Levels of Adequacy of Social Interaction (High, Moderate, Low, Very Low) High Moderate Low Very Low B1 B1 B1 B1 Rural 0.22** 0.21** 0.30** 0.32** 0.01 Semi-rural 0.40*** 0.34*** 0.12 0.05 0.28*** Urban (ref) ref ref ref ref ref Un-stratified Analysis Analysis Stratified by Levels of Adequacy of Social Attachment B1 Settlement (High, Moderate, Low, Very Low) B1 High Moderate Low B1 B1 B1 Very Low B1 Settlement 1 Rural 0.22** 0.09 0.25** 0.39*** 0.18* Semi-rural 0.40*** 0.29** 0.29** 0.17* 0.22*** Urban (ref) ref ref ref ref Ref Standardized and adjusted for sex, religion, occupation, age, education level and physical activity Page 26/26 

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The Open Geography Journal, 2010, 3, 103-114 103 Open Access Radiation and Temperature Responses to a Small Clear-Cut in a Spruce Forest K. Radler*,1, A. Oltchev1,2, O. Panferov1, U. Klinck3 and G. Gravenhorst1 1 Institute of Bioclimatology, Georg-August-University, Büsgenweg 2, D-37077 Göttingen, Germany 2 A.N. Severtsov Institute of Ecology and Evolution of RAS, Leninski Pr. 33, 119071, Moscow, Russia 3 Northwest German Forest Research Station, Department of Environmental Control, Grätzelstr. 2, D-37079 Göttingen, Germany Abstract: Effects of a small clear-cutting on solar radiation, soil and air temperature regimes were investigated by continuous field measurements in a spruce forest in Solling, Central Germany, during vegetation period of 2005. Five meteorological stations, installed in central part of a small clear-cut area (2.5 ha) and close to edges of a surrounding forest, allowed to quantify the spatial variability of meteorological parameters within the clear-cut and to describe the impacts of the forest on clear-cut microclimate. The differences of microclimatic conditions between the clear-cut and the surrounding forest were derived using an additional station installed inside the forest about 150 m from the clear-cut. Results showed that clear-cutting leads to significant changes of spatial and temporal patterns of solar radiation and soil temperature. Solar radiation at the clear-cut was very heterogeneously distributed and about 5-11 times higher than inside the forest. It reached maximum at northeastern part and minimum at southwestern part of the clear-cut. The daily maximal soil temperature at 10 cm depth was measured at northern parts of the clear-cut and it was by up to 6°C higher than in the forest. Daily minimal soil temperature at the clear-cut was about 1-3°C higher than in the forest, too. The main factors influencing the soil temperature patterns were seasonally changed incoming solar radiation, ground vegetation and its phenology, as well as soil moisture. The mean daily maximal air temperature measured at the clear-cut was by up to 2.5°C higher and the mean daily minimal temperature by up to 0.5°C lower than in the surrounded forest. Keywords: Spruce forest, small clear-cut, microclimate, field measurements, solar radiation, soil and air temperature. INTRODUCTION The conversion of Norway spruce (Picea abies) forests has become an important issue for forest management and research in Europe, and transformations of these forests into tree stands mixed with broadleaf species or into uneven-aged forests are aspired. The main objective of such conversions is to increase the ecological stability for those stands, and to make them more resistant against abiotic risks such as storm, snow and ice damage as well as against biotic ones like attacks of fungi and bark beetles. Possible effects of global climate change will most likely enhance such advantages. Moreover, as mixed forests are structurally more divers, they provide more ecological niches for associated fauna, flora and micro-organisms. This in turn improves the suitability of forested landscapes for nature conservation tasks. The conversion of pure spruce stands into mixed forests is also expected to improve the constitution of soils [1]. During the last decade a large part of Norway spruce monocultures in forests of the Lower Saxony of Germany has been converted into stands mixed with beech (Fagus sylvatica) and partially with Douglas fir (Pseudotsuga menziesii). Selective cutting has been used as the main silvicultural method, whereas a sufficient target diameter of *Address correspondence to this author at the Institute of Bioclimatology, Georg-August-University, Büsgenweg 2, D-37077 Göttingen, Germany; Tel: 0049 551 393683; E-mail: kradler1@gwdg.de 1874-9232/10 the trees was considered as main criteria for tree cutting. Recently, in order to accelerate the process of conversion the method of “small clear-cuts”, i.e. 1-3 ha, is considered to be an acceptable alternative [2]. Most likely because leading silviculture paradigms had been favoring other types of forest management, there were surprisingly few experimental studies during the last decades which investigated the changes of micrometeorological conditions after small clear-cuts in a forest. More attention has been paid to effects of large (>10 ha) clear-cuts (e.g. [35]) and on effects of selective thinning (e.g. [6-8]) on forest microclimate. It was shown that such silvicultural methods result in significant changes of solar radiation, air and soil temperature, air humidity, ground surface precipitation, wind speed and direction that vary strongly between the clear-cut, edges and forest interior. The relevant studies related to small clear-cuts usually focused on the ecological consequences of the microclimatic alterations after natural and artificial gap formation, i.e. small openings of less than two tree heights wide (e.g. [911]). Carlson and Groot [12] analyzed microclimatic conditions at small openings of various shapes and sizes up to 1.5 ha within a 19 m tall aspen forest, and found that the irradiance, soil temperature and extremes of air temperature increased with the size of the forest canopy opening. Similar results were obtained by Gray et al. [10] for photosynthetically active radiation (PAR) in gaps of 0.2 to 1.0 tree hights wide within an old-growth forest of Douglas 2010 Bentham Open 104 The Open Geography Journal, 2010, Volume 3 fir and western hemlock. Additionally they showed, that PAR tends to decline within gaps from north via center to south positions, and this heterogeneity increased with gap size; a similar trend was found for the mean soil temperature pattern. Blennow [13] analyzed seasonal patterns of minimal air temperature at a clear-cut area and showed that the spatial pattern of temperature is also attributed to effects of topography which influence drainage, ponding and stagnation of cold air. All these studies complemented quantitatively more comprehensive the general results of classical microclimatological measurements in the forest provided and discussed by Geiger et al. [14]. Radler et al. changed after small clear-cutting (2-3 ha) and 2) How heterogeneous are these changes distributed within the clearcut area? To quantify those changes for the important environmental variables such as solar radiation, soil and air temperature was the main objective of this study. Particularily the latter target, the heterogenity of these parameters within a forest opening has not been in the focus of previous studies. Its quantitative knowledge, however, is obviously useful for a rational management of the regeneration of such areas. For solar radiation is the main source of energy for all life processes in forest ecosystems. It determines thermal and water regimes of ground surface and the atmospheric surface layer and influences transpiration and photosynthesis as the principal processes governing the forest growth. Air and soil temperatures are also key parameters determining the assimilation process of plants, and anomalous very high and low temperature may result in sapling damage or death (e.g. [20, 21]). MATERIALS AND METHODOLOGY Site Description Fig. (1). Aerial photo of study area with locations of the measuring meteorological stations. During the last decade several studies focused on estimation of clear-cutting and thinning effects on wind and turbulent exchange within atmospheric boundary and surface layers (e.g. [15-19]). In relation to forest microclimate such studies are very important because the turbulence regime is one of the main factors determining thermal and moisture status of ground surface and the atmospheric surface layer. The principal results of those field and modeling studies indicate that spatial and temporal wind patterns within the clear-cut are very heterogeneous, and they are strongly influenced by wind speed and direction, as well as by canopy structure and size of the clear-cut area. For assessing the ecological consequences of small clearcuts as compared to the less influencing selective cutting, the Northwest German Forest Research Agency (“Nordwestdeutsche Forstliche Versuchsanstalt - NW-FVA”) has initialized a long-term research program focused on comparisons of different harvesting techniques combined with several types of subsequent regeneration (Douglas fir or beech, artificial vs natural regeneration, different schemes for planting of saplings). The main goal of these studies was to derive general guidelines for the practical forest management, taking into account different influencing factors such as inter- and intra-specific competition of the trees as well as soil and microclimatic conditions. As the latter factor can significantly influence many ecosystem processes, two questions arise: 1) How have microclimatic conditions The study site is located in central part of Germany about 60 km northwest of Goettingen in the Solling highland at 51°46’ N and 09°27’ E, and about 300 m above see level. The clear-cut is 2.6 ha large and has a quadratic form (160m
160m) with diagonals going in south-north and eastwest directions and the forest edges have north-east, northwest, south-west and south-east orientation consequently. The area has a gentle north-western slope with inclination of 6°, so that two western sides are slightly lower than their eastern counterparts. The clear-cut is surrounded from three sides by a 85 year old and 35 m tall pure spruce stand and from south-east side - by a mixed stand of oak, ash and beach trees (Fig. 1). Trees have been felt in winter 2003 by a harvesting machine which also crushed the debris and gathered it at skidding lines. In spring 2004 the saplings of beech and Douglas fir have been planted at the clear-cut area into the bare soil in an experimental lattice of 1 m to 3 m. Vegetation of different herbaceous species has nearly covered the whole area of the clear cut. Along the south-west border a 20 m wide band is partially covered with natural regeneration of spruce of heights between 20 cm and 250 cm. In the southern one-third of the area there are smaller and greater patches covered with great fern (Pteridium aquilinum). The podsolic mineral soil is covered by moder humus with a thickness of about 5 cm. The soil derives from new-red-sandstone, which is covered by a loess layer of up to 1 m. Its main fraction is silt (84%) followed by equal portions of clay and sand (8%). Field Measurements The temporal and spatial patterns of microclimatic conditions at our study area were derived from continuous measurements provided by 6 meteorological stations. Five of those were installed at the clear-cut area (see Fig. 1), one in the centre (Centre) and one - at 12 m distance (1/3 of tree height) to each of four edges (tree lines): north-western (NW), north-eastern (NE), south-western (SW), and southeastern (SE). The sixth station was placed about 150 m to the north-west from the clear-cut within the remained forest and used as a reference (Forest). Each station was equipped with sensors for wind direction and speed, global solar radiation, Radiation and Temperature Responses to a Small Clear-Cut in a Spruce Forest air temperature, relative humidity and precipitation mounted at 2 m above a ground surface, as well as with sensors for soil temperature installed in mineral soil layer at depths of 10 cm and 20 cm. Global solar radiation was measured by means of pyranometers CM3 (Kipp & Zonen, Nederlands), which allow to measure the incoming shortwave radiation within the spectral waveband from 300 to 2500 nm. Air temperature and relative humidity were measured using a platinum resistance thermometer (Pt100) and a capacitive humidity probe in an electrically aspirated shelter (TypeNo. 3030.1000, Theodor Friederichs, Germany) respectively. Soil temperature was measured using a Pt100 sensor installed inside a stainless steel tube of 10 cm length (TypeNo. 2018.0000, Theodor Friederichs, Germany). For data recording and storage the data logger COMBILOG (Theodor Friederichs, Germany) was used. The measurements were sampled with 0.1 Hz frequency and stored as 5-minute averages during the vegetation period of 2005 (from April to October 2005). Estimates of the sky view factor for our measuring sites were based on fisheye-fotos taken by the camera Nikon Coolpix 4500 equipped by lense FC-E8 0.21x with an aperture angle of 180°. Hemispherical photos were analyzed by the WinScanopy software. Data Analysis The temporal and spatial variability of incoming shortwave radiation (R) within the clear-cut area was quantified using 5 minute averaged R (W m-2 s-1), the daily radiation sums R(d) (MJ m-2 day-1), as well as the weekly R(w) and seasonally R(s) averaged R (MJ m-2 day-1) data. To compare radiation regime between the clear-cut and the forest R(d) and R(s) data were used. To quantify shading effects of the surrounding forest on R the ratios Qi of solar radiation at different parts of the clear-cut Ri (i = NW, NE, SW, SE) and at clear-cut center (Centre) were calculated, i.e. Qi= Ri/RCentre To describe the spatial and temporal patterns of soil and air temperature we calculated daily mean Tmean, daily maximal Tmax and minimal Tmin values as well as daily amplitudes A of soil and air temperature. The seasonal averages of these parameters were used for quantifying the differences of the overall seasonal heat gain between measuring sites [20]. To reveal a possible seasonal change of the difference between forest and central part of the clear-cut we considered the differences of the daily maximal
Tmax = TMax_Centre - TMax_Forest and minimal
Tmin = Tmin_Centre TMin_Forest temperature. To quantify the spatial differences of the seasonal dynamics of Tmax and Tmin within the clear-cut, the temperature differences between clear-cut borders Ti (i = NW, NE, SW, SE) and clear-cut center (
Ti = Ti – TCentre) were calculated. Thereby two week averages of Tmin and Tmax were used, in order to reduce the influence of short-term temperature fluctuations. Visual control of data quality, gap filing, data aggregating and analysis were performed by means of SAS software [22]. The Open Geography Journal, 2010, Volume 3 105 RESULTS AND DISCUSSION Solar Radiation Results of the field measurements show that clear-cuting results in drastical increases of solar radiation (Table 1): The highest overall solar irradiance R(s) is observed at the centre of the clear cut, as compaired to the forest site it is elevenfold higher. Shading from surrounding forest stands provides very heterogeneous spatial and temporal distributions of solar radiation within a clear-cut area. The sky view factor for the border measuring sites ranged from 64% to 77% of the centre sky view (Table 1). At the two northern sites of the clear cut about 80 % to 90 % of the irradiance at the central station were measured. Two southern sites of the clear cut receive significantly less solar radiation than the central and northern sites. The seasonal trends of incoming radiation R(d) are characterized by clear maximum in summer months reaching 30 MJ m-2 day-1 at clear-cut centre and 2.6 MJ m-2 day-1 at the forest site (Fig. 2a). The variing conditions of cloudiness cause at the clear-cut the immense day-to-day variability of radiation, which amounted to up to 25 MJ m-2 day-1 vs one tenth of that only in the forest, because of the very low penetration of direct radiation under the forest canopy (e.g. [23]). Apart from short-term variability due to changes of cloudiness, the ratio Qi = Ri/Rcentre (Fig. 2b) shows clear seasonal trends for all clear-cut stations. The highest Q values were always observed at the NE site. They vary around 87% from spring until the middle of August, and in autumn they reach a maximum of close to 100%. The pattern of Q for the NW is quite similar to that of the NE site, however, caused by the smaller sky view factor (Table 1), about 5% smaller. The Q variability at the SW site exhibits the biggest short-term variations with higher values on prevailing cloudy weather conditions and lower ones in periods with dominating sunny weather. It varies around a mean of about 55% until the middle of August, while during the autumn months it shows a stable decreasing trend down to 20%. The seasonal course of Q at the SE site is very close to courses of Q at the northern sites during the period from April to August, while later on it decreases drastically down to 27% in autumn. Table 1. Mean daily (Ri) and Normalized (Qi = Ri/R Centre) Solar Radiation, Measured During the Period from 1 April to 31 October 2005 at Clear-Cut and Forest Sites, and Sky View Factors Derived from Hemispherical Photos (SVi) and Normalized with Sky View at Centre Station (SVQi = SVi/SVCentre) Station Ri (MJ m-2 Day-1) Qi SVi(%) SVQi(%) Centre 13.8 ± 7.0 1.00 80 100 North-West 11.4 ± 5.8 0.83 55 69 North-East 12.1 ± 6.2 0.88 63 79 South-West 6.8 ± 3.9 0.49 53 66 South-East 10.0 ± 5.9 0.72 63 79 Forest 1.2 ± 0.5 0.09 13 16 106 The Open Geography Journal, 2010, Volume 3 Thus, the seasonal pattern of radiation within the clearcut area is characterised by fairly constant and least deviating ratios during summer months and late spring and by an increasing heterogeneity in autumn and early spring. The southern clear-cut parts receive less radiation compared to the open central part, while northern parts get almost the same amount as the center. The main reason for these variations within the clear-cut is the site-specific shading of direct solar radiation by surrounding trees, which with decreasing solar elevation cut off a steadily increasing portion of the daily radiation at southern sites. The chosen experimental clear-cut of Otterbach is somewhat particular because its south-eastern edge is bordered by a deciduous Radler et al. mixed stand. That complicates the spatial and (especially) temporal pattern of solar radiation within the clear-cut since the radiative regime is strongly controlled by structure of the surrounding forest and the latter in turn depends on phenological phase especially for deciduous tree species. To exhibit the reasons for the spatial and temporal heterogeneity of solar radiation regime within the clear-cut area the daily courses of two sunny days were additionally investigated (Fig. 2c). The two selected cloudless days, April 22 and August 18, are characterised by the same day length and sun inclinations, and they were chosen to quantify the effect of varied phenological phases of the surrounding broadleaf trees on solar radiation regime at different parts of Fig. (2a). Seasonal courses of daily solar radiation sums (R) measured by stations in the centre of clear-cut area and at reference station within the forest during the study period from April to October 2005. Fig. (2b). Seasonal course of the ratio Qi = Ri/Rcentre, where Ri ist the daily radiation sum (weekly means) measured at the clear-cut borders (NW, NE, SW, SE), its centre (Centre) and in the forest (Forest). Radiation and Temperature Responses to a Small Clear-Cut in a Spruce Forest The Open Geography Journal, 2010, Volume 3 107 Fig. (2c). Diurnal course of incoming shortwave solar radiation (5 min. averages) at the five clear-cut sites and in the forest for two cloudless days (22th April above and 18th August below). Selected days are characterised by the same day length. the clear-cut area. Comparisons of solar radiation regime at the SE station located closely to the broadleaf forest show that on April 22 the radiation was from sunrise until 11 a.m. much higher and much more variable than on August 18. This difference results from the shading caused by the deciduous forest neighboring the clear-cut from south-east: While in April the buds of trees had been just beginning to burst and the shading was caused by tree stems only, in August all leaves had been fully developed, the canopy is closed and up to 90% of radiation is absorbed by leaves [23]. Therefore, the direct solar radiation, which comes from south-east directions during the morning hours and penetrates through the deciduous stand at the SE border of the clear-cut area, is at the SE measuring site much higher in April than in August. It is also obvious that the shading by stems (in April) causes much higher variability of radiation than the shading by canopy with completely developed folliage (in August). The phenological effect of a steady increment of leaf area in the neighboring deciduous forest stand is visible in Fig. (2b), too, for the trend line of the SE station reveals from April to August a slight but distinct diminishing. With the exception of the reference forest plot, all curves of Fig. (2c) are by about 10-50 W m-2 higher on April 22 than on August 18. To this difference certainly contributes the greater sky view area due to the missing foliage on broadleaf trees, as has been shown by a modelling study on 108 The Open Geography Journal, 2010, Volume 3 Radler et al. this area. Moreover, to the difference observed between the two days could contribute a lower atmospheric transmittance in August, as such effect has been observed at a similar clear-cut area located several kilometres away from the Otterbach study site. Possible reasons for this could be the nearly three times higher content of water vapour, and possibly more airborn particles in the atmosphere. Since higher turbidity of the atmosphere resulted in changes of the ratio between direct and diffuse solar radiation in the total radiation flux, it could have contributed to the somewhat higher solar radiation sum in the forest, because diffuse radiation penetrates better into the forest than direct solar radiation. Apart from this phenological effect Fig. (2c) reveals, that the duration of direct solar radiation differed substantially among our measuring sites on the clear-cut area: for the selected days sunshine duration at the SW site was about 3.5 hours only, as compared to about 7 hours at the SE site. This is probably the most influential difference between the two southern sites. The sunshine duration at the two northern sites was 9 hours, and their daily courses are symmetrical to each other around their maximum at noon. The sunshine duration at central clear-cut site was 11.5 hours for both days. Table 2 shows the daily sums of solar radiation for the selected sunny days as well as two overcast days with nearly the same sun elevation. For the two sunny days the longest duration of direct solar radiation together with the largest sky view area resulted in the biggest radiation gain at centre of the clear-cut. Compared to the forest the ground (vegetation) surface of this site received 30 times more radiation on April 22. The two northern sites received about 15% less, and the SW site nearly 65% less radiation than the central station, on both sampled days. If we assume about 1MJ m-2day-1 less atmospheric transmittance on August 18, then the fully developed leaves caused a reduction of about 3MJ m-2 day-1 or 17% at the SE border of the clear-cut. The solar radiation sums at the two overcast days evinced narrower relations between both the centre and the forest sites, and the southern and the centre sites on the clear-cut. The radiation reduction is least in the forest (ca. 0.5 MJ m-2 day-1 ~ 50%) and largest at the centre of the clear-cut (ca. 19 MJ m-2 day-1 ~ 85%). Table 2. Soil Temperature The properties of the ground surface layer are very heterogeneous on the clear cut, mainly due to heterogeneity of the vegetation/ground cover as well as the structure, composition and moisture of the upper soil layers. Nevertheless a spatial pattern of the soil temperature at 10cm depth was found consistent with the solar irradiance distribution. The centre of the clear cut and the two northern sites show higher values of the mean, the daily maximal and minimal soil temperatures compared to the two southern sites. The soil temperature of the forest site was still lower than of the two southern sites (Table 3). The average daily amplitude was only 0.6°C in the forest, but it ranged from 1.0°C to 1.9°C among the five clear-cut sites. Among the five clear-cut stations the maximal difference for the mean soil temperature was 1.2°C, and for the averaged daily maximal and the minimal values 1.5°C and 1.3°C, respectively. The seasonal course of the daily maximal and minimal temperatures at central part of the clear-cut and at the reference forest site shown in Fig. (3a) indicates that from early spring (the beginning of measuring campaign) to the hottest summer period in July/August the soil temperature increased by 13°C at clear-cut sites and by 9°C at the reference forest plot. The highest soil temperature, which had been observed in late July, was about 20°C at the clearcut and nearly 16°C in the forest. From May to September the daily minimum of soil temperature at the clear-cut was always higher than the daily maximum in the forest. The differences between clear-cut and forest ranged for the maximal temperature (
Tmax = TMax_Centre - TMax_Forest) from 0.0°C to 4.5°C and for the minimal temperature (
Tmin = TMin_Center - TMin_Forest) from -1.0°C to 3.2°C. Peak values occurred for
Tmax in May and July, and for
Tmin from late May to the beginning of August. In seasonal course
Tmax was always up to 2°C larger than
Tmin. The seasonal course of the daily amplitudes A (Fig. 3b) exhibits heterogeneous trends in spring, and for all stations a decreasing trend from the middle of May to the end of the vegetation period. For clear-cut stations these patterns could be influenced by ground vegetation cumulating in the clearcut area from May to October. It intercepts solar radiation during daytime and reduces the long-wave emission from the Comparison of daily Sums of Solar Radiation R i (MJ m -2 day-1) Measured for 4 Selected Days in Spring and Summer (i.e. ~2 Months Before and After Summer Solstice) Characterized by Cloudless and Overcast Sky Conditions. Qi = Ri/RCentre (See Fig. 2c) April Station 22th (Cloudless) August 19th (Overcast) 18th (Cloudless) 20th (Overcast) Ri Qi Ri Qi Ri Qi Ri Qi Centre 23.7 1.00 5.3 1.00 22.2 1.00 3.1 1.00 North-West 19.9 0.84 4.1 0.77 18.9 0.85 2.4 0.78 North-East 20.0 0.84 4.4 0.86 18.7 0.84 2.6 0.86 South-West 9.1 0.38 3.6 0.68 8.0 0.36 2.2 0.71 South-East 18.9 0.80 4.8 0.91 14.7 0.66 2.6 0.86 Forest 0.8 0.03 0.5 0.09 1.3 0.06 0.7 0.21 Radiation and Temperature Responses to a Small Clear-Cut in a Spruce Forest soil surface during the night [24]. The smallest A and the lowest variation of A were observed at the reference forest station. Among the clear-cut sites the NW site had the smallest A throughout the study period. It ranged from 0.4°C to 1.6°C, and it was very similar to the A course at the Central station, however, always about 1°C lower. The A courses at NE and SW sites were quite similar during the measuring period, and differed by less than 0.5°C. The highest seasonal variability of A was observed at SE site, where it reached its maximum of 3°C in late May, while in summer and autumn it decreased to values of less than 1°C. The reason for such trend is probably the variability of solar radiation reaching the soil surface, which is influenced, on the one hand, by weather conditions and, on the other hand, by phenological changes in the neighboring deciduous forest (see previous section) and by the dense vegetation cover of fern species growing around the measuring plot. Table 3. Mean Daily Tmean, Daily Maximal Tmax, Daily Minimal Tmin (0:00-8:00) Soil Temperature (°C) at 2m a.g.l., and their Daily Amplitude A = Tmax - Tmin with Standard Deviations for Five Clear-Cut and the Forest Reference Sites Averaged for Measuring Period from April to October 2005 Station/Site Tmean Tmax Tmin A Clear-cut Centre 13.6 ± 3.0 14.6 ± 3.0 12.7 ± 3.1 1.9 ± 0.8 North-West 13.4 ± 2.8 13.9 ± 2.7 12.9 ± 2.8 1.0 ± 0.5 North-East 13.6 ± 3.3 14.5 ± 3.4 12.8 ± 3.2 1.7 ± 0.8 South-West 12.4± 3.3 13.1 ± 3.4 11.6 ± 3.3 1.5 ± 0.7 South-East 12.6 ± 2.8 13.4 ± 2.8 11.8 ± 3.0 1.6 ± 1.1 Forest 11.1 ± 2.6 11.4 ± 2.6 10.8 ± 2.6 0.6 ± 0.3 The Open Geography Journal, 2010, Volume 3 109 The seasonal course of the spatial patterns of maximal and minimal soil temperature within the clear-cut shows (Fig. 3c), that the differences
Ti = Ti – TCentre between clearcut center and clear-cut borders (NW, NE, SW, SE) changed significantly during the measuring period. They ranged between +1.5°C and -3.0°C for the daily maximal, and between +1.0°C and -2.0°C for the daily minimal values. Since the value of maximal soil temperature is mainly determined by incoming solar radiation absorbed by ground surface, their seasonal course should have a trend similar to the radiation pattern. However, although the incoming solar radiation in clear-cut center is larger than at clear-cut borders, the maximal soil temperature at the NE site is up to 1.2°C higher than at clear-cut centre during June and July. Such difference could be explained by lower turbulent exchange in the vicinity of the surrounding forest as has been shown by several modelling studies (e.g. [16, 18]). It may also be explained by a stronger reduction of soil moisture at this part of the clear-cut resulting in lower thermal conductivity of the upper soil layers, and here indicated by a larger daily temperature amplitude (see Fig. 3b) of the upper soil layer (0-10cm). At the SW site the seasonal course of the deviation (from centre site) seems to follow the trend of the relative radiation gain at this station (see Fig. 2b), i.e. least deviating around summer solstice, and more deviating in spring and autumn for both maximal and minimal temperatures. At the SE site a similar effect seems to be involved in the period of late spring, but in the following month of July the maximal soil temperature exhibits a steep relative decrease to a level that was 2.0-2.5°C lower than the temperature at the Centre site. This is most likely influenced by a reduced radiation gain due to foliage development in the neighboring deciduous forest. In addition, the soil surface around our measuring sensor at SE site was covered by Fig. (3a). Seasonal course of daily maximum and minimum of soil temperature (T) measured by stations in the centre of clear-cut area and within the forest for study period from April to October 2005. 110 The Open Geography Journal, 2010, Volume 3 Radler et al. Fig. (3b). Seasonal course of mean daily amplitude (two weeks averaging) of soil temperature at the five measuring stations on the clear-cut area and at the reference forest station during the study period from April to October 2005. Fig. (3c). Seasonal course of mean daily maxima (above) and minima (below) of soil temperature at all measuring sites during the study period from April to October 2005. Values are plotted as deviations from the respective value at the central station
Ti = Ti – TCentre. Radiation and Temperature Responses to a Small Clear-Cut in a Spruce Forest dense ground vegetation of a fern species (Pteridium aquilinum), which started foliating in late June and absorpted increasingly more of the incoming solar radiation in August and September. The daily minimal temperature mainly depended on weather conditions of the night, but was also strongly influenced by temperature conditions of the previous day. Thus, their seasonal dynamics is very similar to the course pattern of the maximal temperature for all clear-cut sites (Fig. 3c). At the southern sites minimal soil temperature is always, up to 2.0°C, lower than at the clear-cut centre. At the NW site minimal soil temperature is quite similar to values at clear-cut centre during the summer period, while it is always larger than in clear-cut centre during spring and autumn. At NE site
Tmin has a seasonal trend, which is very similar to the trend of
Tmax. Air Temperature The results of provided measurements show a small spatial variability of the air temperature within the clear-cut as well as a little difference between the clear-cut and the reference forest site as compaired to soil temperature patterns. The seasonally averaged temperatures (Table 4) differed between the clear-cut centre and the reference forest plot by 0.3°C only. For the daily maximal temperature this difference is more significant (+1.7°C) while the difference of daily minimal values is negative and relatively small (-0.3°C) too. The mean daily amplitude of air temperature is at the clear-cut centre 2°C larger than at the forest site. The corresponding temperature range among the five clear-cut sites was of the same order of magnitude, i.e. 0.6°C for the daily mean, 1.2°C for daily maximal Tmax, and 0.4°C for daily minimal temperature Tmin. Variability of daily amplitude A within the clear-cut sites does not exceed 1°C. The pattern of average Tmax mirrors generally the radiation and soil temperature pattern, i.e. at the central and the northern sites it is higher than at both the southern and the forest sites. Table 4. Mean Daily Tmean, Daily Maximal Tmax, Daily Minimal Tmin (0:00-8:00) Air Temperature (°C) at 10 cm Depth, and their Daily Amplitude A = Tmax Tmin with Standard Deviations for Five Clear-Cut and the Forest Reference Sites Averaged for Measuring Period from April to October 2005 Station/Site Tmean Tmax Tmin A Clear-cut Centre 13.7 ± 4.5 19.2 ± 5.6 9.3 ± 4.1 9.9 ± 3.5 North-West 13.7 ± 4.6 18.9 ± 5.6 9.4 ± 4.2 9.5 ± 3.5 North-East 13.8 ± 4.5 19.2 ± 5.6 9.5 ± 4.1 9.7 ± 3.6 South-West 13.4 ± 4.5 18.0 ± 5.2 9.3 ± 4.2 8.7 ± 3.0 South-East 13.2 ± 4.4 18.5 ± 5.5 9.1 ± 4.1 9.4 ± 3.4 Forest 13.4 ± 4.4 17.5 ± 5.2 9.6 ± 4.2 7.9 ± 3.0 The seasonal course of the air temperatures (Fig. 4a-c) reveals no trend for the daily amplitude and for Tmin, but for Tmax an increasing difference between northern and southern sites from spring to autumn. The maximal difference The Open Geography Journal, 2010, Volume 3 111 between the clear-cut area and the reference forest site was 4.4°C and it was observed at the NE station on sunny day of August 29. Maximal air temperature in the central part of the clear-cut reached 26.0°C on this day, and the global radiation was 20.5 MJ m-2day-1. The values of minimal air temperature (Fig. 4c) were, however, even on clear nights unexpectedly similar at all measuring sites. This could be caused by a horizontal downhill air flow on the clear-cut and into the trunk space of the surrounding spruce forest. The Tmin was at clear-cut sites usually somewhat lower than at the forest site. The SE station tended to show the lowest night temperature, and the greatest difference was observed at the SW station on the clear night of May 20, when minimal air temperature was 2.1°C lower than in the forest. Such differencies may be explained by structure of radiation and energy balances at the clear-cut area and at the reference forest plot. The radiation balance of the clear-cut area (i.e. surfaces of soil and plants) is always positive during the day and negative during night-time. The surface gets more energy than it looses during the day and it looses more energy by long-wave emission than it gains during the night. The surface and the air in 2m height at the clear-cut should get warmer during daytime and cooler during night time as compared to the surface (of soil and understorey) and air conditions within the forest, where the radiation balance is much reduced. The daily maximum air and surface temperatures are, therefore, lowest in the forest and highest on the clear-cut while this is reversed for the daily minimum, as our measurement results confirmed (Table 4 and Fig. 4c). CONCLUSIONS The results of the field study in the small clear-cut area in Otterbach showed that the changing sun position during the course of a day and a year resulted in a quite heterogeneous solar irradiance field on the clear cut with higher values at the northern sides differenciated by beech phenology and by the ratio of diffuse sky irradiance to direct solar irradiances. The soil (10 cm depth) temperature is distributed in a similar pattern, which is diverging where it is influenced by dead and active vegetation cover and by soil moisture. The temperature of the 10 cm soil layer varied most strongly in time and space, because it depends on direct irradiance, on soil heat conductivity and capacity as well as on vegetation cover and its phenology. An increase of up to 4.5°C during sunny days certainly influences several ecological processes like for instance the nutrient cycling (e.g. [9, 25, 26]). The spatial range of 2°C for the daily maximal soil temperature within our clear-cut of 2-3 ha fits well into findings of similar measurements in gaps (e.g. [10]), although direct compairisons are difficult because measuring depths as well as sizes of forest openings differed. This spatial heterogenity contributes to a niche differentiation with species tolerating hotter and temporarily drier conditions in the centre and at northern borders of the clear-cut. Only about half as wide ranged the air temperature on the clear cut area (<1°C) and between the clear cut and the neighboring spruce stand (<2°C). This is mainly due to the standard meteorological measuring hight of 2m (a.g.l.). For the moving air aggregates the different heat sources at the 112 The Open Geography Journal, 2010, Volume 3 Radler et al. Fig. (4a). Seasonal course of daily maximum and minimum of air temperature (T) measured by stations in the centre of clear-cut area and within the forest for study period from April to October 2005. Fig. (4b). Seasonal course of mean daily amplitude (two weeks averaging) of air temperature at five measuring stations on the clear-cut area and at the reference forest plot during the study period from April to October 2005. ground surface and levels the temperature rapidly by vertical turbulence and horizontal advection. Thus this pattern would have been more heterogeneous at a measuring hight of 20cm a.g.l. For the forest management the main results of this case study may be useful in several ways: (1) They provide hints for the planning to decrease the risk of sapling mortality and to maximize the success of forest regeneration with this silviculture strategy. If, as in our case, the target is a mixed forest stand of Douglas fir and beech, these two species could be allocated according to radiation and/or soil temperature gradients rather than randomly or regularily on such small clear-cuts. (2) In the context of an ecosystem management it provides one of most rare examples for measuring the magnitude of environmental spatial gradients, which are relevant for other management objectives, e.g. providing habitat for wildlife species or forest arthropods (see e.g. [27, 28]). (3) Last but not least such results are accordingly valid in all types of disturbances of compairable size, and Radiation and Temperature Responses to a Small Clear-Cut in a Spruce Forest The Open Geography Journal, 2010, Volume 3 113 Fig. (4c). Seasonal course of mean daily maxima (above) and minima (below) of air temperature at the sites within the clear-cut area and at the reference forest plot during the study period from April to October 2005. Values are plotted as deviations from the respective value at the central station
Ti = Ti – TCentre. independant of causes like wind throws, forest fires or bark beetle impacts. Forschungsgemeinschaft (German Research Foundation), Grant No. Gr 738/16-2. ACKNOWLEDGEMENTS REFERENCES We thank D. Fellert for the complete installation and his committed maintenance of the microclimate stations, H. Kreilein and H. Schäfer for contributing their technical experiences. This study was supported by Deutsche [1] [2] Spiecker H, Ed. Norway Spruce conversion - options and consequences. Leiden: European Forest Institute 2004. Röhrig E, Bartsch N, von Lüpke B. Waldbau auf ökologischer Grundlage. 7th ed. Stuttgart: Ulmer 2006. 114 The Open Geography Journal, 2010, Volume 3 [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] Radler et al. Stathers RJ, Black TA, Novak MD. Modelling soil temperature in forest clearcuts using climate station data. Agric For Meteorol 1985; 36: 153-64. Chen J, Franklin JF, Spies TA. 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Eur J Forest Res 2007; 126: 1-9. Revised: October 26, 2009 Accepted: October 26, 2009 © Radler et al.; Licensee Bentham Open. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. 

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International Journal of Molecular Sciences Article Comparative Physiological and Transcriptome Analyses of Tolerant and Susceptible Cultivars Reveal the Molecular Mechanism of Cold Tolerance in Anthurium andraeanum Na Dou 1 , Li Li 1 , Yifu Fang 2 , Shoujin Fan 1 1 2 * Citation: Dou, N.; Li, L.; Fang, Y.; Fan, S.; Wu, C. Comparative Physiological and Transcriptome Analyses of Tolerant and Susceptible Cultivars Reveal the Molecular Mechanism of Cold Tolerance in Anthurium andraeanum. Int. J. Mol. Sci. 2024, 25, 250. https://doi.org/10.3390/ ijms25010250 and Chunxia Wu 1, * Shandong Provincial Key Laboratory of Plant Stress, College of Life Sciences, Shandong Normal University, Wenhua East Road 88, Jinan 250014, China; fansj@sdnu.edu.cn (S.F.) Institute of Ornamental Plants, Shandong Provincial Academy of Forestry, Wenhua East Road 42, Jinan 250010, China; fyf7741@163.com Correspondence: cxwu1001@sdnu.edu.cn Abstract: Anthurium andraeanum is a tropical ornamental flower. The cost of Anthurium production is higher under low temperature (non-freezing) conditions; therefore, it is important to increase its cold tolerance. However, the molecular mechanisms underlying the response of Anthurium to cold stress remain elusive. In this study, comparative physiological and transcriptome sequencing analyses of two cultivars with contrasting cold tolerances were conducted to evaluate the cold stress response at the flowering stage. The activities of superoxide dismutase and peroxidase and the contents of proline, soluble sugar, and malondialdehyde increased under cold stress in the leaves of the cold tolerant cultivar Elegang (E) and cold susceptible cultivar Menghuang (MH), while the soluble protein content decreased in MH and increased in E. Using RNA sequencing, 24,695 differentially expressed genes (DEGs) were identified from comparisons between cultivars under the same conditions or between the treatment and control groups of a single cultivar, 9132 of which were common cold-responsive DEGs. Heat-shock proteins and pectinesterases were upregulated in E and downregulated in MH, indicating that these proteins are essential for Anthurium cold tolerance. Furthermore, four modules related to cold treatment were obtained by weighted gene co-expression network analysis. The expression of the top 20 hub genes in these modules was induced by cold stress in E or MH, suggesting they might be crucial contributors to cold tolerance. DEGs were significantly enriched in plant hormone signal transduction pathways, trehalose metabolism, and ribosomal proteins, suggesting these processes play important roles in Anthurium’s cold stress response. This study provides a basis for elucidating the mechanism of cold tolerance in A. andraeanum and potential targets for molecular breeding. Keywords: Anthurium andraeanum; cold stress; transcriptome; WGCNA; plant hormone signal; trehalose; ribosomal protein Academic Editor: Michael Moustakas Received: 19 November 2023 Revised: 16 December 2023 Accepted: 21 December 2023 Published: 23 December 2023 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/). 1. Introduction Plants are sessile organisms, and during their life cycle, they encounter several abiotic stresses caused by harsh climatic changes. Cold stress is a detrimental abiotic stress affecting the growth and development, reducing the productivity and quality, and limiting the geological distribution of plants [1–3]. For example, low temperature stress can lead to plasma membrane peroxidation caused by reactive oxygen species (ROS), thus causing damage to internal cell structures [4,5]. To cope with cold stress, plants have developed complex strategies, including changes in a variety of molecular, physiological, and biochemical pathways [6]. During exposure to cold, the activities of antioxidant enzymes such as superoxide dismutase (SOD) and peroxidase (POD) and the contents of proline, soluble sugars, and proteins change; therefore, they are considered as physiological indicators of cold response in plants [7,8]. Genotypes with higher amounts of these compounds usually show cold resistance [9,10]. Int. J. Mol. Sci. 2024, 25, 250. https://doi.org/10.3390/ijms25010250 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2024, 25, 250 2 of 28 Sugars play an essential role in plant response to cold stress. Trehalose, a nonreducing disaccharide, has been detected in a wide range of organisms, including bacteria, fungi, invertebrates, and plants [11]. It not only serves as an energy source and a protectant but also functions as a signaling molecule in plants [12,13]. Trehalose is only present in trace amounts under optimal conditions, and its production increases in response to stresses [13,14]. In plants, trehalose is synthesized from UDP-glucose and glucose 6-phosphate in a reaction catalyzed by trehalose-phosphate synthase (TPS), and trehalose-phosphate phosphatase (TPP)and is degraded into glucose by trehalase [11]. Overexpression of TPS or TPP can improve the stress tolerance of transgenic plants [15,16]. Trehalose-6-phosphate (T6P), the precursor of trehalose biosynthesis in plants, is also a signaling molecule and interacts with SUCROSE-NON-FERMENTING1-RELATED KINASE1 (SnRK1) to balance sucrose levels in plants [17,18]. Numerous studies have demonstrated that plant hormones play important roles in regulating signal transduction in response to cold stress [2,19]. The plant hormone abscisic acid (ABA) is essential for inducing stomatal closure, seed dormancy, and abiotic stress response [20]. Cold stress can increase the endogenous ABA levels in plants, leading to increased cold resistance [21,22]. There are also ABA-independent signaling pathways involved in the response to low temperature stress. For example, cold stress affects the transport of auxin and inhibits the intracellular trafficking of auxin efflux carriers [23]; this in turn affects organogenesis and morphogenesis, which are regulated by the accumulation and polar distribution of auxin in plant tissues [24]. Other phytohormones, such as brassinosteroid (BR) [25], jasmonic acid (JA) [26], ethylene [27], and salicylic acid (SA) [28], also play a critical role in plant cold stress response. Low temperature also induces a change in gene transcription and protein synthesis. For example, the expression pattern of ribosomal proteins changes during plant response to low temperature, thereby affecting mRNA translation and protein synthesis. The accumulation of three soybean ribosomal proteins (GmRPS13, GmRPS6, and GmRPL37) was found to be induced by low temperature [29]. Plastid ribosomal proteins, which are essential components of protein synthesis machinery, also have diverse roles in plant growth, development, and response to abiotic stress, especially temperature stress [30]. For example, chilling stress was found to cause ribosomal pausing during photosystem II component-D1 protein elongation in tomato [31]. Rpl33, a ribosomal protein that does not affect plant viability and growth under standard conditions, is required for sustaining plastid translation under cold stress [32]. Loss of function of the ribosomal subunit protein S5 (RPS5), a plastid 30S subunit, was found to affect photosynthesis proteins and cold stress response, possibly via a reduction in plastid translational capacity [33]. Plants with mutations in TCD11, which encodes RPS6 in chloroplasts, grow normally at 32 ◦ C but die at 20 ◦ C, indicating that TCD11 functions in chloroplast development at low temperature [34]. All these findings suggest that cold stress has the potential to impact virtually all plant processes by affecting protein translation. Anthurium andraeanum (hereafter Anthurium) is a tropical ornamental plant known for its colorful spathe (i.e., leaf-like bracts enclosing the flowers) and spadix (inflorescence containing small flowers). Anthurium is commercially produced worldwide to be sold as cut flowers or as a potted plant. It does not grow well when the temperature decreases below 12 ◦ C [35]. Therefore, Anthurium is often grown in greenhouses in north and northeast China, and supplemental heating is necessary in late autumn, winter, and early spring, which increases energy consumption and production costs. Therefore, it is of great interest to elucidate the molecular mechanism of Anthurium’s response to cold stress, to enable the development of cold-tolerant Anthurium varieties. Studies aimed at elucidating such mechanisms have identified genes potentially involved in the response of Anthurium to cold stress. De novo transcriptome analysis of 3-month-old Anthurium cv. Alabama seedlings subjected to 6 ◦ C temperature revealed a number of cold-inducible transcription factors and pathways in Anthurium [36]. Thereafter, AnAPX [35], AabHLH35 [37], and microRNA158 [38] were found to function in the response Int. J. Mol. Sci. 2024, 25, 250 3 of 28 to cold stress. In addition, exogenous application of chemical agents, γ-aminobutyric acid (GABA) [39], and putrescine [40] have been shown to alleviate chilling injury in Anthurium. Although these reports have provided insight into cold tolerance in Anthurium, the molecular mechanisms underlying the response to low temperature stress are not clear and need to be further investigated. In a preliminary study (unpublished data), the cultivars of Anthurium plants were collected from a producer in Shandong Province, China, and the cold responses of these plants were evaluated. Two cultivars, one cold-tolerant and the other cold-sensitive, were selected for further study. In this study, the morphological and physiological changes of these two cultivars when exposed to cold conditions were assessed. Then, the transcriptome profiles of the two cultivars under cold stress at the full flower phase were determined. The cultivar-specific genes and pathways and those common to both were revealed from analysis of the transcriptome data. This study provides insight into the mechanisms underlying Anthurium’s cold-stress response, thus facilitating the development of coldtolerant Anthurium varieties. 2. Results 2.1. Cold Stress Triggers Morphological Differences between E and MH To compare cold responses among different Anthurium cultivars, we monitored the growth of 16 Anthurium cultivars at different temperatures as a preliminary study (unpublished data). Two Anthurium cultivars, Elegang (E, cold-tolerant) and Menghuan (MH, cold-susceptible), were selected to further examine the differences in the responses of Anthurium cultivars to cold stress. These cultivars are the most popular flowers in the Chinese market. When the Anthurium plants were subjected to 6 ◦ C for 3 days, portions of the E spathes became deep red, but there was no visible change in the color of MH spathes (Figure 1B,D, 6 ◦ C). When the plants were grown for 3 days at 4 ◦ C, the E spathes were all deep red (Figure 1B, 4 ◦ C), while the petioles of some young leaves of MH began to soften and the leaves began to droop (Figure 1C, red arrow). These phenotypic results show that MH is more susceptible to cold stress than E. 2.2. Cold Stress Affects the Physiological Responses of E and MH To further evaluate the cold tolerance of these two Anthurium cultivars, the physiological responses were measured in the leaves of control and cold-treated plants. The activities of SOD and POD and the contents of osmo-protectants (proline, soluble sugar, and soluble protein) at 25/22 ◦ C (control conditions) were higher in MH than in E (Figure 2A,B,D,E,F). The change trends in SOD activity, POD activity, and the contents of proline and soluble sugars in response to cold treatment were similar in the two cultivars, but the levels of these parameters in E were lower than those in MH (Figure 2A,B,D,E). Cold stress induces ROS accumulation in plant cells, and antioxidative enzymes are the key ROS scavengers [41]. The activities of SOD and POD increased in both cultivars under cold stress (Figure 2A,B). However, the POD activity in MH was almost three-fold higher at 6 ◦ C than at 25/22 ◦ C, though the level at 4 ◦ C was the same as that at the control temperature (Figure 2B). The content of MDA, which is an important marker of lipid peroxidation and membrane damage [42,43], increased about 16-fold in MH at 6 ◦ C compared with the control and was higher than that of E after cold treatment (Figure 2C), indicating that E had a higher capacity to cope with oxidative stress than MH. Osmoprotectants including proline, soluble sugars, and soluble proteins accumulate in cells to protect against cell damage [44]. The proline content increased in response to cold stress, and in both cultivars it did not significantly differ between the 6 ◦ C and 4 ◦ C treatments (Figure 2D). The soluble sugar contents were higher at 6 ◦ C compared with the control for both varieties, but those in MH were 4.8-fold higher while those in E were only 2.2fold higher (Figure 2E). No significant difference was observed in soluble sugar content between the control and the treatment at 4 ◦ C (Figure 2E). Interestingly, the soluble protein content of MH decreased under cold stress relative to the control, while that of E increased Int. J. Mol. Sci. 2024, 25, 250 4 of 28 Int. J. Mol. Sci. 2024, 25, 250 4 of 27 (Figure 2F). Consistent with the phenotypic results, the physiological data indicated that MH is sensitive to cold stress and E is cold-tolerant. Int. J. Mol. Sci. 2024, 25, 250 5 of 27 higher (Figure 2E). No significant difference was observed in soluble sugar content beFigure 1.the Phenotypic responses of Eofand MH cold stress. Images of E and grown at 25/22 °C ◦C tween control and the treatment at MH 4to°C 2E). Interestingly, the soluble protein Figure 1. Phenotypic responses E and to(Figure cold stress. Images of E MH and MH grown at 25/22 (control) then keptdecreased under theunder same conditions orrelative transferred to control, 6 °C or◦ 4while °C for◦that 3 days. (A,C) Side content of MH cold stress to the of E increased (control) then kept under the same conditions or transferred to 6 C or 4 C for 3 days. (A,C) Side views of the and (B,D) topthe views of the plants. The red indicates data the softened petiole (Figure 2F).plants Consistent with phenotypic results, thearrow physiological indicated that ofviews MH. of the plants and (B,D) top views of the plants. The red arrow indicates the softened petiole MH is sensitive to cold stress and E is cold-tolerant. of MH. 2.2. Cold Stress Affects the Physiological Responses of E and MH To further evaluate the cold tolerance of these two Anthurium cultivars, the physiological responses were measured in the leaves of control and cold-treated plants. The activities of SOD and POD and the contents of osmo-protectants (proline, soluble sugar, and soluble protein) at 25/22 °C (control conditions) were higher in MH than in E (Figure 2A,B,D,E,F). The change trends in SOD activity, POD activity, and the contents of proline and soluble sugars in response to cold treatment were similar in the two cultivars, but the levels of these parameters in E were lower than those in MH (Figure 2A,B,D,E). Cold stress induces ROS accumulation in plant cells, and antioxidative enzymes are the key ROS scavengers [41]. The activities of SOD and POD increased in both cultivars under cold stress (Figure 2A,B). However, the POD activity in MH was almost three-fold higher at 6 °C than at 25/22 °C, though the level at 4 °C was the same as that at the control temperature (Figure 2B). The content of MDA, which is an important marker of lipid peroxidation and membrane damage [42,43], increased about 16-fold in MH at 6 °C compared with the control and was higher than that of E after cold treatment (Figure 2C), indicating that E had a higher capacity to cope with oxidative stress than MH. Osmoprotectants including physiological proline, soluble sugars,ofand soluble accumulate in cellsdismutase to Figure 2. Differential responses E and MH toproteins cold stress. (A) Superoxide protect against cell damage [44]. The proline content increased in response to cold stress, Figure Differential physiological responses of E and cold stress. (A) Superoxide dismutase (SOD) 2.activity, (B) peroxidase (POD) activity, andMH the to contents of (C) malondialdehyde (MDA), and in both cultivars it did not significantly differ between the 6 °C and 4 °C treatments (SOD) activity, (B) peroxidase (POD) activity, and the contents of (C) malondialdehyde (MDA), (D) (D) proline, (E) soluble sugar, and (F) soluble protein. Error bars represent SD (n = 3). The statistical (Figure 2D). The soluble contents higher atbars 6 °C compared with theThe control for proline, (E) soluble sugar,sugar andby (F) soluble were protein. = 3). significance was determined multiple t-tests. pError < 0.05, *; prepresent < 0.01, **;SD p <(n0.001, ***; pstatistical < 0.0001, ****. both varieties, those in by MH were 4.8-fold those E were 2.2-fold significance wasbut determined multiple t-tests. p <higher 0.05, *;while p < 0.01, **; p <in0.001, ***; only p < 0.0001, ****. 2.3. RNA Sequencing and De Novo Transcriptome Assembly Bliss et al. (2012) estimated the genome size of Anthurium to be 4.6 Gb based on the bulk nuclear DNA contents for 77 accessions determined by flow cell cytometry [45]. To date, no genome sequence of A. andraeanum has been reported. Through de novo tran- Int. J. Mol. Sci. 2024, 25, 250 5 of 28 2.3. RNA Sequencing and De Novo Transcriptome Assembly Bliss et al. (2012) estimated the genome size of Anthurium to be 4.6 Gb based on the bulk nuclear DNA contents for 77 accessions determined by flow cell cytometry [45]. To date, no genome sequence of A. andraeanum has been reported. Through de novo transcriptome analysis, genetic information for organisms can be obtained without genomic information [46], novel genes regulating pathways can be discovered, and the expression patterns can be elucidated. Eighteen cDNA libraries were constructed using the RNA extracted from the leaves of E (cold-tolerant) and MH (cold-sensitive) incubated at 25/22 ◦ C (E_CK and MH_CK), 6 ◦ C (E_6 and MH_6), and 4 ◦ C (E_4 and MH_4) for 3 days. The libraries were sequenced on an Illumina Novaseq™ 6000. Sequencing generated a total of 129.44 Gb of raw reads for all 18 samples and an average of 7.19 Gb of reads for each sample (Table S1). After processing, 117.67 Gb of the clean reads were generated with an average Q30 of 95.09% (Table 1). The Trinity assembler generated 132,108 unigenes with a minimum length of 201 bp (Table 1, Figure 3A). The lengths of unigenes varied from 201 bp to 16,127 bp with an average length of 648 bp and an N50 length of 1207 bp, and the GC content was 44.84% (Table 1, Figure 3A). The clean reads were assembled into 290,721 transcripts with lengths ranging from 200 bp to 16,127 bp (Table 1). Table 1. Assembly statistics for the A. andraeanum transcriptome. Assembly Statistics Raw reads (average) Total number of clean reads Clean reads (average) Clean bases (G) Q20% (average) Q30% (average) GC% (average) Number of transcripts (>200 bp) Number of unigenes Minimum length (bp) Maximum length (bp) Average length (bp) N50 length (bp) GC content (%) Total assembled bases 47,943,471.56 840,806,398 46,711,466.56 117.67 98.48 95.09 51.42 290,721 132,108 201 16,127 648 1207 44.84 85,547,816 2.4. Functional Annotation of Unigenes To determine their possible functions, the 132,108 unigenes were annotated using six databases (NCBI Nr, SwissProt, Gene Ontology [GO], Kyoto Encyclopedia of Genes and Genomes [KEGG], Pfam, and eggNOG). A total of 34,390 unigene sequences had matches in the NCBI Nr database (26.03%), 30,479 in eggNOG (23.07%), 23,730 in GO (17.96%), 23,270 in Pfam (17.61%), 20,586 in KEGG (15.58%), and 19,112 in SwissProt (14.47%), with the most matches in the NCBI Nr database (Figure 3B). The unigenes annotated in the Nr database can reflect the species distribution statistics. The Anthurium unigene sequences showed the highest similarity to sequences from Elaeis guineensis (13.1%), followed by Phoenix dactylifera (12.35%), Vitis vinifera (4.91%), Cinnamomum micranthum (4.64%), Nelumbo nucifera (4.55%), and Musa acuminata (3.77%) (Figure 3C). In the eggNOG database, the annotated unigenes were divided into 23 categories (Figure 3D). “Replication, recombination, and repair” (3833, 12.58%), “posttranslational modification, protein turnover, chaperones” (1951, 6.40%), and “signal transduction mechanisms” (1668, 5.47%) were the top three categories in terms of numbers of annotated genes. The categories with the fewest unigenes were “cell motility” (6, 0.020%) and “nuclear structure” (1, 0.003%). Int. J. Mol. Sci. 2024, 25, 250 6 of 28 (A) (B) Figure 3. Cont. Int. J. Mol. Sci. 2024, 25, 250 7 of 28 (C) (D) Figure 3. Overview of RNA sequencing results and annotation of unigenes. (A) Distribution of unigenes. (B) Overlap between the number of all unigenes according to six databases. (C) Distribution of unigene annotations based on the Nr database for the species distribution statistics. (D) eggNOG functional classification of all unigenes. To analyze the functions of the unigenes, the GO database annotations were classified in three main ontologies: biological process (BP, 19,884, 83.79%), molecular function (MF, 20,472, 86.27%), and cellular component (CC, 21,085, 88.85%) (Table S2). Unigenes were 2 annotated to 4810 BP terms, and the term with the most annotations was “regulation of transcription, DNA-templated” (1365, 6.86%). For CC, “nucleus” (6677, 31.67%) and “cytoplasm” (3945, 18.71%) were the most abundant among the 970 terms, while for MF, “protein Int. J. Mol. Sci. 2024, 25, 250 8 of 28 binding” (2665, 13.02%) and “ATP binding” (1731, 8.46%) were the two most frequent terms (Figure S1). A total of 20,586 unigenes was clustered into 20 KEGG pathway categories (Figure S2). The most significant KEGG pathway was “carbohydrate metabolism” (1623, 7.88%), followed by “translation” (1500, 7.29%) and “folding, sorting, and degradation” (1346, 6.54%) (Figure S2). 2.5. Differential Expression Analysis of Anthurium Genes To dissect the different cold-stress responses of the two Anthurium cultivars at the transcript level, DEGs were identified between cultivars under control (CK) and cold-stress conditions (4 or 6 ◦ C) using the criteria |log2 FC| > 2 and FDR < 0.001. A total of 8829 DEGs were identified between E and MH: 4070 DEGs were detected from the comparison of E_CK and MH_CK (1682 up and 2388 down), 2755 DEGs from E_6 and MH_6 (1474 up and 1281 down), and 4769 DEGs from E_4 and MH_4 (2600 up and 2169 down) (Figures 4A and S3A, Table S3). These results indicated that many genes are differentially expressed between the two cultivars under control and cold-stress conditions. Gene expression differences in both cultivars between control vs. cold (6 ◦ C and 4 ◦ C treatments) were calculated. A total of 22,305 DEGs were obtained from comparisons 6 ◦ C versus CK and 4 ◦ C versus CK for both cultivars: 14,188 (8616 up and 5572 down) and 5861 (3203 up and 2658 down) DEGs were identified between 6 ◦ C and CK for E and MH, respectively, and 14,079 (8166 up and 5913 down) and 13,516 (6880 up and 6636 down) DEGs were identified between 4 ◦ C and CK for E and MH, respectively (Figures 4B and S3B, Table S4). There were more upregulated genes than downregulated genes in all comparisons (Figure S3B). At 6 ◦ C, there were more DEGs in the cold-tolerant cultivar E than in the cold-sensitive cultivar MH (Figure S3B), which indicated that more genes show changes in expression to response to cold in E than in MH in the early stage of cold stress. The 22,305 DEGs were assigned to two groups based on the overlap between comparisons: (1) genotype-specific cold-stress-responsive (CSR) DEGs differentially expressed between the control and cold treatment in only one cultivar and (2) common CSR DEGs differentially expressed in both cultivars. There were 2126 and 1842 DEGs in the E_6 vs. E_CK and E_4 vs. E_CK comparisons, respectively, and there were 3981 DEGs common to E_6 vs. E_CK and E_4 vs. E_CK. Thus, 7949 DEGs were specific to the cold-tolerant cultivar E (Figure 4B). Similarly, there were 394 and 3616 DEGs in the MH_6 vs. MH_CK and MH_4 vs. MH_CK comparisons, respectively, and there were 1214 DEGs common to MH_6 vs. MH_CK and MH_4 vs. MH_CK. Thus, 5224 DEGs were specific to the cold-sensitive cultivar MH. There were 9132 (100, 290, 56, 277, 3323, 207, 499, 3592, 788) common CSR DEGs identified in the 6 ◦ C vs. CK and 4 ◦ C vs. CK comparisons for both cultivars (Figure 4B, Table S5). The identification of common CSR DEGs indicat that there are conserved cold-stress responses in the two cultivars. However, further analysis of these genes showed that the expression patterns of these genes were inconsistent between the two cultivars under , and 4 ◦ C stress (Figure 4C). These common DEGs were further analyzed to investigate the differences in their expression patterns between the two cultivars. Among these 9132 common CSR DEGs, when comparing 6 ◦ C with CK, there were 8081 DEGs (4504 upregulated and 3577 downregulated genes) in E and 4695 DEGs (2520 upregulated and 2175 downregulated genes) in MH (Figure 4D); whereas, at 4 ◦ C, there were 8256 DEGs (4490 upregulated and 3766 downregulated genes) in E and 8686 DEGs (4641 upregulated and 4045 downregulated genes) in MH (Figure 4E). Among the common CSR DEGs, more genes were upregulated under cold stress than downregulated. Int. J. Mol. Sci. 2024, 25, 250 Int. J. Mol. Sci. 2024, 25, 250 8 of 27 the two cultivars under 6 °C and 4 °C stress (Figure 4C). These common DEGs were further analyzed to investigate the differences in their expression patterns between the two cultivars. 9 of 28 Figure 4. two cultivars in response to cold (A) Venn showingshowing DEGs’ profile Figure 4.DEGs DEGsofof two cultivars in response to stress. cold stress. (A)diagram Venn diagram DEGs’ profile between E and MH under different cold treatments. (B) Venn diagram showing the overlap in DEGs between E and MH under different cold treatments. (B) Venn diagram showing the overlap in DEGs in both cultivars between cold and control conditions. (C–E) The 9132 common DEGs differentially in both cultivars between cold and control conditions. (C–E) The 9132 common DEGs differentially expressed in both cultivars during cold stress. (C) Heatmap showing the results of clustering analexpressed both cultivars stress.diagram (C) Heatmap showing the results ofexpressed clustering analysis ysis of the in 9132 common CSRduring DEGs.cold (D) Venn showing the overlap in DEGs of the69132 common CSR DEGs. (D) Venn diagram showing thethe overlap after °C cold stress in both cultivars. (E) Venn diagram showing overlapininDEGs DEGsexpressed expressed after 6 ◦ C understress 4 °C cold stress in both cultivars. cold in both cultivars. (E) Venn diagram showing the overlap in DEGs expressed under 4 ◦ C cold stress in both cultivars. Among these 9132 common CSR DEGs, when comparing 6 °C with CK, there were 8081 All DEGs and 3577 downregulated genes) E and 4695 DEGs (2520 the(4504 CSRupregulated DEGs common to both varieties and bothincold treatments were compared. upregulated and 2175 downregulated genes) in MH (Figure 4D); whereas, at 4 °C, there ◦ When the Anthurium plants were treated at 6 C for 3 days, 2156 DEGs were upregulated were 8256 DEGs (4490 upregulated and 3766 downregulated genes) in E and 8686 DEGs and 1935 DEGs were downregulated exclusively in E, while 161 and 544 DEGs were specif(4641 upregulated and 4045 downregulated genes) in MH (Figure 4E). Among the comically upregulated and downregulated in MH, respectively (Figure 4D). However, 4 DEGs mon CSR DEGs, more genes were upregulated under cold stress than downregulated. wereAll upregulated in Ecommon and downregulated in and MH,both while 15treatments DEGs were downregulated the CSR DEGs to both varieties cold were comin E and upregulated in MH. A total of 2344 DEGs were upregulated while 1627 DEGs pared. When the Anthurium plants were treated at 6 °C for 3 days, 2156 DEGs were upwere downregulated in were both downregulated E and MH. Under the 4 ◦in C E, treatment, were upregregulated and 1935 DEGs exclusively while 161290 andDEGs 544 DEGs ulated and 56 DEGs were downregulated only in E, while 460 DEGs were upregulated and 316 DEGs were downregulated only in MH (Figure 4E). Twenty-seven DEGs were upregulated in E and downregulated in MH, and eight DEGs showed the opposite expression patterns. Furthermore, 4173 and 3702 DEGs were upregulated and downregulated, respectively, in both cultivars. Under 6 ◦ C stress, there were more DEGs specific to E than to MH, while at 4 ◦ C there were fewer E-specific DEGs and significantly more up- and down-regulated DEGs in both cultivars compared with the 6 ◦ C treatment. These results indicate that the gene expression changes enable the plant to cope with cold stress occurred at an earlier stage in E than in MH. Int. J. Mol. Sci. 2024, 25, 250 10 of 28 2.6. Functional Analysis of Common CSR DEGs in Anthurium To characterize the functions of the DEGs, we performed GO and KEGG enrichment analysis using OmicStudio (https://www.omicstudio.cn/tool, accessed on 14 October 2023) for the DEG groups described above (Figures 5, S4 and S5). Under 6 ◦ C stress, DEGs (2156 up and 1935 down) specific to the cold-tolerant cultivar E were enriched in the GO terms “secondary metabolite biosynthesis”; “oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, NAD(P)H as one donor, and incorporation of one atom of oxygen”; and “extracellular region”. Interestingly, some CoA synthases for long-chain fatty acid stood out significantly. KEGG analysis revealed enrichment in “pentose and glucuronate interconversions”, “fatty acid elongation”, and “phenylalanine metabolism”. For the cold-sensitive cultivar MH, the specific DEGs (161 up and 544 down) were enriched in the GO terms “extracellular region”, “regulation of organ growth”, and “plant-type hypersensitive response” as well as the KEGG pathways “plant hormone signal transduction”, “alpha-linolenic acid metabolism”, and “MAPK signaling pathway—plant”. This result indicated that changes in energy metabolism and membrane lipids occur in the cold-tolerant cultivar E, whereas signal transduction is affected in the cold-sensitive cultivar MH. After 4 ◦ C stress, E-specific DEGs (290 up and 56 down) were enriched in “maintenance of protein location in nucleus”, “bundle sheath cell fate specification”, and “radial pattern formation” as well as the KEGG pathways “alpha-linolenic acid metabolism”, “biotin metabolism”, and “galactose metabolism”. The MH-specific DEGs (460 up and 316 down) were enriched in the GO terms “chloroplast”, “chloroplast thylakoid membrane”, “chloroplast stroma”, “NAD(P)H dehydrogenase complex (plastoquinone)” and “chloroplast thylakoid lumen”, which are the cellular components associated with photosynthesis. The enriched KEGG pathways were “carbon fixation in photosynthetic organisms”, “carotenoid biosynthesis”, and “nitrogen metabolism” (Figure 5). The enrichment of these categories suggests that photosynthesis and the carbon and nitrogen balance may be affected in MH. These results reflect differences in the responses of the two Anthurium varieties to cold stress. DEGs in both varieties may reflect common mechanisms in the response of Anthurium to cold stress (Figure S5). Under 6 ◦ C stress, the 2344 DEGs upregulated in both cultivars were enriched in trehalose-related terms (GO:0004805, GO:0003825, GO:0070413, and GO:0005992), “response to chitin”, “DNA-binding transcription factor activity”, and transcription-related terms. For the downregulated DEGs common to both cultivars, photosynthesis-related biological process and cellular component terms were enriched. Under 4 ◦ C stress, the common upregulated DEGs in both cultivars were enriched in “response to chitin”, “sequence-specific DNA binding”, “regulation of defense response”, “protein phosphorylation”, and trehalose-related terms. For downregulated DEGs, the enriched terms were “cell wall organization”, “extracellular region”, and photosynthesisrelated biological process and cellular component terms. The E-specific DEGs at 6 ◦ C were also enriched in CoA synthase for long-chain fatty acid. These results suggest that trehalose metabolism is important in the response of Anthurium to cold stress and that photosynthesis-related processes are affected under cold stress. Among the common CSR DEGs, four DEGs were upregulated in E and downregulated in MH under 6 ◦ C stress (Figure 4D). One was a heat-shock protein, which is involved in protein processing in the endoplasmic reticulum (Table 2). These four DEGs may be critical in the response of E to cold stress. Fifteen DEGs were upregulated in MH and downregulated in E at 6 ◦ C (Table 2). Three encoded a glutamate receptor (GLR), three encoded subtilisin-like proteases (SBTs), two encoded cysteine-rich receptor-like protein kinases (CRK), one encoded a BURP-domain containing protein, one encoded a zinc-finger protein, and one encoded transmembrane protein. After 4 ◦ C treatment, there were 27 DEGs upregulated in E and downregulated in MH, encoding one heat-shock protein (HSP), one heat-stress transcription factor (HSF), three cell-wall-associated enzymes (pectinesterase or pectinesterase-like proteins), one putative nucleotidyltransferase, one fatty acid amide hydrolase-like (FAAH), one MYB transcription factor, and one chloroplastic early light- Int. J. Mol. Sci. 2024, 25, 250 Int. J. Mol. Sci. 2024, 25, 250 11 of 28 induced protein (ELIP1) (Table 3). The eight DEGs downregulated in E and upregulated in MH encoded GPAT6, a probable disease resistance protein, CRK, and CYC P450 (Table 3). of 27 These common CSR DEGs with the opposite expression patterns may 10 play vital roles in Anthurium cold tolerance. Their functions need to be further studied to determine if they can be used as target genes to improve Anthurium cold tolerance. Figure 5. Gene Ontology enrichment analysis of different groups of the 9132 common CSR DEGs. Figure 5. Gene Ontology enrichment analysis of different groups of the 9132 common CSR DEGs. Int. J. Mol. Sci. 2024, 25, 250 12 of 28 Table 2. The common CSR-DEGs with opposite expression patterns in the cultivars E and MH under 6 ◦ C stress. Four Common CSR-DEGs Upregulated in E but Downregulated in MH under 6 ◦ C Stress Gene_ID TRINITY_DN43394_c2_g5 TRINITY_DN56057_c0_g5 TRINITY_DN42951_c1_g2 TRINITY_DN52943_c0_g2 Name log2 FC (E_6 vs. E_CK) log2 FC (MH_6 vs. MH_CK) HSP17.4B 3.57 2.68 2.85 3.55 −4.21 −3.36 −2.61 −2.42 Annotation uncharacterized protein LOC110667824 [Hevea brasiliensis] 17.4 kDa class III heat-shock protein [Phoenix dactylifera] Fifteen Common CSR-DEGs Upregulated in MH but Downregulated in E under 6 ◦ C Stress TRINITY_DN43741_c0_g2 - −3.28 4.19 TRINITY_DN35319_c2_g1 - −2.04 3.91 TRINITY_DN52379_c0_g2 TRINITY_DN39241_c0_g1 TRINITY_DN47085_c1_g1 TRINITY_DN42045_c2_g2 TRINITY_DN35790_c0_g1 TRINITY_DN37319_c0_g3 TRINITY_DN50258_c0_g4 BURP3 SBT1.8 SBT1.6 SBT1.9 COL16 GLR2.1 - −3.76 −2.19 −2.05 −2.60 −5.34 −4.19 −2.25 9.79 4.83 4.74 8.07 3.72 4.13 4.23 TRINITY_DN55351_c1_g2 CRK28 −4.24 4.48 TRINITY_DN32295_c0_g1 CRK6 −3.10 4.38 TRINITY_DN54550_c0_g1 At3g47200 −2.38 3.13 TRINITY_DN56023_c0_g2 TRINITY_DN56023_c0_g1 TRINITY_DN49565_c0_g3 GLR2.2 GLR2.8 - −2.30 −2.03 −3.55 6.76 6.54 3.72 hypothetical protein L195_g017092, partial [Trifolium pratense] uncharacterized protein LOC18094378 isoform X3 [Populus trichocarpa] BURP domain-containing protein 3 [Phoenix dactylifera] hypothetical protein B296_00025367 [Ensete ventricosum] hypothetical protein B296_00025367 [Ensete ventricosum] subtilisin-like protease SBT1.9 [Phoenix dactylifera] uncharacterized protein LOC111300501 [Durio zibethinus] PREDICTED: glutamate receptor 2.7-like [Elaeis guineensis] hypothetical protein Ahy_B09g100047 isoform B [Arachis hypogaea] cysteine-rich receptor-like protein kinase 8 [Phoenix dactylifera] PREDICTED: UPF0481 protein At3g47200-like [Elaeis guineensis] hypothetical protein AQUCO_03100069v1 [Aquilegia coerulea] Glutamate receptor 2.8 [Vitis vinifera] - Table 3. The common CSR DEGs with opposite expression patterns in cultivars E and MH under 4 ◦ C stress. Twenty-Seven Common CSR-DEGs Upregulated in E but Downregulated in MH under 4 ◦ C Stress Name log2 FC (E_4 vs. E_CK) log2 FC (MH_4 vs. MH_CK) Annotation TRINITY_DN45412_c0_g4 - 4.33 −4.71 TRINITY_DN56876_c2_g1 TRINITY_DN43394_c2_g5 TRINITY_DN52540_c1_g2 TRINITY_DN56279_c2_g2 TRINITY_DN46551_c2_g2 TRINITY_DN35567_c1_g1 TRINITY_DN37079_c1_g3 TRINITY_DN36186_c0_g3 TRINITY_DN36954_c1_g7 TRINITY_DN56438_c1_g1 TRINITY_DN51698_c0_g1 TRINITY_DN51173_c0_g1 TRINITY_DN33920_c1_g3 TRINITY_DN54886_c1_g3 TRINITY_DN37706_c0_g1 TRINITY_DN53485_c0_g2 TRINITY_DN54221_c0_g4 TRINITY_DN42775_c1_g1 TRINITY_DN50024_c0_g1 PME1 HSFA2B TIP4-3 FAAH PME1 MPE3 - 2.59 4.86 3.65 2.11 2.65 3.09 6.35 2.79 3.14 5.30 2.95 2.56 2.87 2.74 3.18 4.42 2.09 2.80 2.61 −3.07 −3.71 −4.19 −3.64 −3.66 −9.03 −3.61 −4.52 −2.65 −2.24 −6.30 −3.04 −3.55 −2.06 −4.45 −3.28 −2.37 −3.45 −2.82 TRINITY_DN42193_c0_g1 - 2.66 −2.58 TRINITY_DN54864_c0_g4 TRINITY_DN42951_c1_g2 TRINITY_DN51579_c0_g3 TRINITY_DN45042_c0_g3 TRINITY_DN52943_c0_g2 TRINITY_DN44614_c2_g1 HSP17.4B ELIP1 2.61 2.13 2.18 2.06 4.06 5.51 −2.85 −2.54 −2.27 −3.21 −2.12 −2.03 PREDICTED: uncharacterized protein LOC105048415 [Elaeis guineensis] pectinesterase 3 [Jatropha curcas] PREDICTED: heat shock factor protein HSF30-like [Elaeis guineensis] putative nucleotidyltransferase, Ribonuclease H [Rosa chinensis] PREDICTED: probable aquaporin TIP4-3 [Elaeis guineensis] fatty acid amide hydrolase-like [Ananas comosus] pectinesterase 3 [Manihot esculenta] pectinesterase 1-like [Dendrobium catenatum] PREDICTED: uncharacterized protein LOC105803676 [Gossypium raimondii] transcription factor MYBS3-like isoform X2 [Quercus suber] hypothetical protein AQUCO_05400137v1 [Aquilegia coerulea] 17.4 kDa class III heat-shock protein [Phoenix dactylifera] chloroplastic early light-induced protein [Crocus sativus] Gene_ID Eight Common CSR-DEGs Downregulated in E but Upregulated in MH under 4 ◦ C Stress TRINITY_DN46057_c0_g4 GPAT6 −4.12 7.57 TRINITY_DN55055_c2_g2 At1g61180 −4.25 3.37 TRINITY_DN44362_c1_g2 TRINITY_DN49073_c0_g2 TRINITY_DN55351_c1_g2 CRK28 −2.86 −2.71 −3.46 3.86 2.24 3.54 TRINITY_DN39795_c1_g3 - −2.66 2.25 TRINITY_DN45091_c1_g1 TRINITY_DN51899_c2_g3 CYP704C1 - −2.29 −4.76 5.97 7.31 Glycerol-3-phosphate 2-O-acyltransferase 6 [Ananas comosus] probable disease resistance protein At5g63020 isoform X1 [Citrus sinensis] hypothetical protein Ahy_B09g100047 isoform B [Arachis hypogaea] PREDICTED: uncharacterized protein LOC109022142, partial [Juglans regia] PREDICTED: cytochrome P450 704C1-like [Elaeis guineensis] - Int. J. Mol. Sci. 2024, 25, 250 tightly associated with MH_6 (R = 0.83, p = 2 × 10−5) and MH_4 (R = 0.89, p = 1 × 10−6), respectively, and both modules were negatively correlated with the E samples. 13 of 28 In the turquoise module, the top enriched GO terms were “nucleus”, “plasma membrane”, and “cytoplasm” and the top KEGG pathways were “plant hormone signal transduction”, “plant-pathogen interaction”, and “MAPK-signaling pathway-plant”. The top 2.7. Co-Expression Network of DEGs enriched GO terms for theAnalysis greenyellow module were “extracellular exosome”, “memWeighted gene co-expression analysis (WGCNA) a systems biology method brane”, and “structural constituentnetwork of ribosome”. The top threeisenriched KEGG pathways for describing the“oxidative correlations among genes across [47]. WGCNA is usedThese to not were “ribosome”, phosphorylation”, and samples “glycolysis/gluconeogenesis”. only construct gene networks but also detect gene modules and identify the central players results indicat that signal transduction, plant immunity genes, protein synthesis, and deg(i.e., hubare genes) withinfor modules. To to comprehensively themodule, mechanism cold radation important resistance cold stress in E.decipher In the tan thereofwere response in Anthurium, all DEGs (24,695) were used to build a gene co-expression network. three top GO terms with the same DEGs and Q-values: “quercetin 3-O-glucosyltransferase Via sample clustering, 18 samples were included in and the “flavonoid analysis. Based on pairwise activity”, “quercetin 7-O-glucosyltransferase activity”, glucuronidation”. correlations of gene expression, 16 modules were identified (marked with different in The top enriched KEGG pathways were “carotenoid biosynthesis”, “pentose andcolors glucuFigure interconversions”, 6A). A correlation heatmap of module-cold was constructed to clearly ronate and “MAPK signaling relationships pathway—plant”. The top three GO visualize the correlation of modules and cold treatment (Figure 6B). The turquoise module terms for the yellow module were “nucleus”, “cytoplasm”, and “mitochondrion”, while wastop associated with the were E_6 (R“sphingolipid = 0.76, p = 3 metabolism”, × 10−4 ), and the greenyellow module was the KEGG pathways “starch and sucrose metabo− 5 associated with E_4 (R = 0.79, p = 8 × 10 ). Importantly, these two modules were negatively lism”, and “amino sugar and nucleotide sugar metabolism” (Figure S6, Table S6). These associated with the MH samples. The tan and yellow modules were tightly associated with findings indicate that carotenoid biosynthesis and sugar metabolism may be involved in MH_6 (R = 0.83, p = 2 × 10−5 ) and MH_4 (R = 0.89, p = 1 × 10−6 ), respectively, and both the response of MH to cold stress. modules were negatively correlated with the E samples. Figure6.6.Identification Identification co-expression gene modules using DEGs. (A) hierarchical The hierarchical cluster Figure ofof co-expression gene modules using DEGs. (A) The cluster tree tree shows 16 co-expressed modules identified by WGCNA. Different modules are marked with shows 16 co-expressed modules identified by WGCNA. Different modules are marked with different colors.colors. (B) Module-cold treatment correlation. Each row a module. Each Each column cordifferent (B) Module-cold treatment correlation. Eachrepresents row represents a module. column responds to a specific sample. The color of each cell at the row–column intersection indicates corresponds to a specific sample. The color of each cell at the row–column intersection indicatesthe the correlation correlationcoefficient coefficientbetween betweenthe themodule moduleand andsample. sample.Red Redindicates indicatesaahigh highdegree degreeof ofcorrelation correlation between betweenaaspecific specificmodule moduleand andthe thesample. sample. The topturquoise 20 hub genes werethe identified by theGO cytohubba plug-in using the MCC algoIn the module, top enriched terms were “nucleus”, “plasma memrithm in Cytoscape software. The edges with a weight value no less than 0.2 were used to brane”, and “cytoplasm” and the top KEGG pathways were “plant hormone signal transcalculate top hub gene. In the turquoise TIFY 10, MAN3, At1g32860 (glucan duction”,the “plant-pathogen interaction”, andmodule, “MAPK-signaling pathway-plant”. The top endo-1,3-beta-glucosidase 14-like), AOS1 (allenewere oxide synthase 1, exosome”, chloroplastic-like), TL1 enriched GO terms for the greenyellow module “extracellular “membrane”, (thaumatin-like 1), and (receptor-like protein ANXUR1) were included and “structuralprotein constituent of FER ribosome”. The top three kinase enriched KEGG pathways were in the top 20“oxidative hub genesphosphorylation”, (Table S7). All these except FER, have been These found results to be “ribosome”, and genes, “glycolysis/gluconeogenesis”. involved in abiotic biotic stress responses in plants the green-yellow modindicat that signal and transduction, plant immunity genes,[48–52]. protein For synthesis, and degradation are the important for resistance to cold stress in E. Inproteins, the tan module, theredehydrogenase were three top ule, top 20 hub genes encoded nine ribosome three NADH GO termsthree with the same DEGs and two Q-values: “quercetin 3-O-glucosyltransferase activity”, subunits, elongation factors, cytochrome oxidase subunits, an ATP synthase “quercetin activity”,(Table and “flavonoid glucuronidation”. The top subunit, and7-O-glucosyltransferase fructose-bisphosphate aldolase S7). This result indicates that protein enriched KEGG pathways were “pentose and glucuronate inand ATP synthesis are related to “carotenoid the responsebiosynthesis”, to cold. For the tan and yellow modules, terconversions”, and “MAPK signaling pathway—plant”. The top three GO terms for the yellow module were “nucleus”, “cytoplasm”, and “mitochondrion”, while the top KEGG pathways were “sphingolipid metabolism”, “starch and sucrose metabolism”, and “amino sugar and nucleotide sugar metabolism” (Figure S6, Table S6). These findings indicate that Int. J. Mol. Sci. 2024, 25, 250 14 of 28 carotenoid biosynthesis and sugar metabolism may be involved in the response of MH to cold stress. The top 20 hub genes were identified by the cytohubba plug-in using the MCC algorithm in Cytoscape software. The edges with a weight value no less than 0.2 were used to calculate the top hub gene. In the turquoise module, TIFY 10, MAN3, At1g32860 (glucan endo-1,3-beta-glucosidase 14-like), AOS1 (allene oxide synthase 1, chloroplasticlike), TL1 (thaumatin-like protein 1), and FER (receptor-like protein kinase ANXUR1) were included in the top 20 hub genes (Table S7). All these genes, except FER, have been found to be involved in abiotic and biotic stress responses in plants [48–52]. For the green-yellow module, the top 20 hub genes encoded nine ribosome proteins, three NADH dehydrogenase subunits, three elongation factors, two cytochrome oxidase subunits, an ATP synthase subunit, and fructose-bisphosphate aldolase (Table S7). This result indicates that protein and ATP synthesis are related to the response to cold. For the tan and yellow modules, most of the top 20 hub genes were not annotated in the NCBI database (Table S7). Three Asp1 genes (encoding aspartic proteinases) were hub genes in the tan module (Table S7). The co-expression networks of these hub genes were visualized using Cytoscape (Figure 7). The top genes in the turquoise and greenyellow modules were remarkably upregulated in the E_6 or E_4 samples, while those in the tan and yellow modules had significantly high levels of expression in MH_6 and MH_4, respectively (Figure 7). 2.8. Plant Hormone Signal Transduction Is Involved in the Cold Stress Response in Anthurium Plant hormones play important roles in plant growth and plant response to stress [53]. In this study, there were 226 DEGs between treatment and control annotated to plant hormone signal transduction (Table S8), indicating that cold stress activates various plant hormone pathways (e.g., auxin, cytokinin, ethylene, ABA, JA, SA, BR, and GA) in Anthurium leaves (Figure 8, Table S8). The expression profiles of these 226 DEGs are shown in Figure 8. The auxin pathway genes encoding AUX1 and TIR1 were downregulated. Only 4 of the 12 AUX/IAA DEGs were upregulated. DEGs related to SAUR were all downregulated in MH, and the DEGs that were upregulated in response to cold in E showed no difference in MH. In both cultivars, negative regulators of the GA pathway were downregulated, and the downstream target genes were upregulated. All ethylene pathway DEGs, except ETR, were upregulated in E but downregulated in MH. There were more upregulated BR and JA pathway DEGs in E than in MH, and the number of DEGs encoding MYC2 and JAZ were especially high. The distinct expression patterns of these DEGs in E and MH reveal a complicated mechanism involving phytohormones under cold stress. Among the eight plant-hormone-related DEGs, most were related to auxin (54 DEGs), BR (42 DEGs), and JA (34 DEGs). Moreover, most of these DEGs were upregulated after cold stress. This suggests that low temperature induces the upregulation of genes related to phytohormones, to protect against cold injury and confer tolerance to cold stress in Anthurium. Int. J. Mol. Sci. 2024, 25, 250 15 of 28 gure 7 Heatmap (A) turquoise: (B) greenyellow: (C) tan: (D) yellow: Figure 7. Identification of hub genes of the four key modules responsive to cold stress in Anthurium. The co-expression network of the top 20 hub genes of the key modules was visualized by Cytoscape. The heatmaps show the expression of the top genes of the four modules. (A) The turquoise module. (B) The greenyellow module. (C) The tan module. (D) The yellow module. The hub genes with the highest connectivity to other genes are shown in the nodes. The node colors corresponds to connectivity values, and their sizes represent the module membership value calculated by WGCNA. The darker and larger the node, the greater the hubness of the gene. The width of the lines between nodes corresponds to weight as calculated by WGCNA. The wider the line, the higher the weight value between the nodes. The values in the heatmap represent the z-score of the transcript levels in different samples. Red indicates a high expression level, while green is low. Int. J. Mol. Sci. 2024, 25, 250 Int. J. Mol. Sci. 2024, 25, 250 16 of 27 16 of 28 Figure 8. The expression patterns of DEGs in the “plant hormones signal transduction” pathway. Figure 8. The expression patterns of DEGs in the “plant hormones signal transduction” pathway. Heatmap shows the expression of DEGs associated with different hormones in E and MH. In Heatmap shows the expression of DEGs associated with different hormones in E and MH. the In the pathways, blueblue indicates the the downregulated genes, orange indicates downandand upregulated DEGs, pathways, indicates downregulated genes, orange indicates downupregulated DEGs, andand redred indicates thethe upregulated genes. Mean TPM values of the three replicates were used to to indicates upregulated genes. Mean TPM values of the three replicates were used represent gene expression value in the heatmap. represent gene expression value in the heatmap. 2.9.2.9. Trehalose-Related Genes AreAre Induced in Anthurium in Response to Cold Stress Trehalose-Related Genes Induced in Anthurium in Response to Cold Stress Trehalose is aisnonreducing disaccharide found in diverse organisms andand serves as an Trehalose a nonreducing disaccharide found in diverse organisms serves as an energy source as well as an osmolyte and/or protein/membrane protectant [11].[11]. Trehalose energy source as well as an osmolyte and/or protein/membrane protectant Trehalose andand its its precursor, T6P, areare involved in plant responses to multiple abiotic stresses [11].[11]. precursor, T6P, involved in plant responses to multiple abiotic stresses TPP enzymes in trehalose synthesis, while trehalase is the enzyme TPSTPS andand TPP areare thethe keykey enzymes in trehalose synthesis, while trehalase is the enzyme responsible degradation of trehalose. In this study, trehalose-related terms responsible for for thethe degradation of trehalose. In this study, trehalose-related GOGO terms were significantly enriched in upregulated common DEGs (Figure S5).found We found were significantly enriched in upregulated common CSRCSR DEGs (Figure S5). We 25 25 trehalose-related (22 TPS, 1 trehalase) were upregulated under cold trehalose-related DEGsDEGs (22 TPS, 2TPP,2TPP, and and 1 trehalase) thatthat were upregulated under cold Int. J. Mol. Sci. 2024, 25, 250 Int. J. Mol. Sci. 2024, 25, 250 17 of 27 17 of 28 stress (Figure (Figure9,9,Table Table S9). Most of them upregulated in E under cold but stress but stress S9). Most of them werewere upregulated in E under cold stress hardly hardly or lowly expressed in the control and MH samples (Figure 9). or lowly expressed in the control and MH samples (Figure 9). Figure metabolism. Mean TPM values of Figure 9. 9. The The expression expressionpatterns patternsofof2525DEGs DEGsinvolved involvedinintrehalose trehalose metabolism. Mean TPM values the three replicates were used to represent thethe gene expression value in the heatmap. TheThe values in of the three replicates were used to represent gene expression value in the heatmap. values the heatmap represent the the z-score of the levels in different samples. Red indicates a higha in the heatmap represent z-score of transcript the transcript levels in different samples. Red indicates high expression expression level, level, whilewhile blue isblue low.is low. 2.10. 2.10. Ribosomal Ribosomal Proteins Proteins Are Are Involved Involved in in Anthurium’s Anthurium’s Response Response to to Cold Cold Stress Stress Ribosomes are the primary sites of translation and protein synthesis, Ribosomes are the primary sites of translation and protein synthesis, and and their their main main components are protein and rRNA. In the greenyellow module, ribosome was components are protein and rRNA. In the greenyellow module, ribosome was the the top top enenriched KEGG pathway.Seventy-eight Seventy-eightDEGs DEGsencoding encodingribosomal ribosomalproteins proteins were were found found to riched KEGG pathway. to be differentially expressed in response to cold (Figure 10A, Table S10). These DEGs showed be differentially expressed in response to cold (Figure 10A, Table S10). These DEGs distinct expression patterns in the two cultivars under cold treatment (Figure 10B,C). showed distinct expression patterns in the two cultivars under cold treatment (Figure There were more upregulated DEGs than downregulated DEGs, and there were more 10B,C). There were more upregulated DEGs than downregulated DEGs, and there were upregulated DEGs in E than in MH and more downregulated DEGs in MH than in E. more upregulated DEGs in E than in MH and more downregulated DEGs in MH than in Moreover, 25 upregulated DEGs were only found in the E_4 vs. E_CK comparison. These E. Moreover, 25 upregulated DEGs were only found in the E_4 vs. E_CK comparison. results suggest that mRNA translation and protein synthesis are more active in E (coldThese results suggest that mRNA translation and protein synthesis are more active in E resistant) than in the cold-sensitive variety MH, which is consistent with the differences in (cold-resistant) than in the cold-sensitive variety MH, which is consistent with the differphysiological indexes between these varieties. ences in physiological indexes between these varieties. Int. J. Mol. Sci. 2024, 25, 250 Int. J. Mol. Sci. 2024, 25, 250 18 of 27 18 of 28 Figure10. 10.The The expression patterns of DEGs encoding ribosome proteins (map03010). (A)riboThe Figure expression patterns of DEGs encoding ribosome proteins (map03010). (A) The somal DEGs.DEGs. Blue indicates down-regulated genes, genes, orangeorange indicates down- and upregulated DEGs, ribosomal Blue indicates down-regulated indicates downand upregulated and redand indicates upregulated genes. (B) Heatmap of DEGs large-subunit ribosomal proDEGs, red indicates upregulated genes. (B) Heatmap of encoding DEGs encoding large-subunit ribosomal teins. (C) Heatmap of DEGs encoding small-subunit ribosomal proteins. In the heatmap, mean TPM proteins. (C) Heatmap of DEGs encoding small-subunit ribosomal proteins. In the heatmap, mean values of the three samples were used to represent the gene expression value. TPM values of the three samples were used to represent the gene expression value. Int. J. Mol. Sci. 2024, 25, 250 19 of 28 2.11. Validation of DEGs by qRT-PCR To validate the RNA-seq results, 16 common DEGs were randomly selected for qRTPCR analysis. The qRT-PCR data were generally consistent with the RNA-seq data, which confirmed the authenticity of the DEGs in this study (Figure S7). 3. Discussion Low-temperature stress is detrimental and affects the growth and development, productivity, and distribution of plants. Anthurium is a tropical ornamental plant that is sold as cut flowers or potted plants. It is popular for its brightly colored spathe and spadix, so research has mainly focused on the mechanism giving rise to Anthurium’s spathe color and the environmental factors that affect color. There have been few studies on Anthurium’s response to cold stress, and the mechanism remains unclear. Tian et al. performed a time-series (1 h, 5 h, 24 h) transcriptome analysis of 3-month-old Anthurium cv. Alabama under 6 ◦ C conditions [36]. Some transcription factors and pathways that may be involved in response to cold stress were identified. AnAPX gene cloned from Anthurium cv. Alabama that was upregulated under 6 ◦ C stress was previously shown to enhance cold tolerance by preventing cell membrane damage under chilling stress [35]. AabHLH35 was identified from transcriptome analysis of a dark-green leaf color mutant of Anthurium and was shown to improve the cold tolerance of transgenic Arabidopsis [37]. MicroRNAs have also been shown to be involved in regulating plant responses to cold stress. Aa-miR158 was found to be expressed at higher levels in Anthurium under cold stress and to improve cold tolerance when overexpressed in Arabidopsis [38]. In this study, two Anthurium cultivars, E (coldtolerant) and MH (cold-susceptible), were used as experimental materials. Transcriptome sequences were used to characterize the molecular mechanism of cold responses in Anthurium. The DEGs common to the different cultivars were analyzed, and the key modules and hub genes were identified through WGCNA. 3.1. Morphological and Physiological Changes Differ between E and MH under Cold Stress Low temperatures influence plant growth and development by causing various morphophysiological and biochemical changes [54,55]. In this study, the spathe color (red) of E was deeper after cold stress. For MH, the petiole of young leaves softened, and the leaf drooped (Figure 1). Therefore, E is resistant and MH is sensitive to cold stress. Low-temperature stress causes metabolic imbalance and inhibits various cellular processes. Under cold stress, the photostatic imbalance induces cell lipid peroxidation and damage to the membrane structure, thereby influencing cell membrane fluidity and producing MDA, which is often used as a marker of lipid peroxidation and membrane damage [42,43]. The leaf MDA content in MH was higher than that of the control and of E after cold treatment (Figure 2C). These results indicate that the cell membrane in MH is damaged more severely than that in E under cold stress. Antioxidant enzymes such as SOD and POD and osmolytes such as proline, soluble sugar, and soluble protein, usually play a key role in balancing ROS and the osmotic gradient in the cell [41,56]. Here, the activities of SOD and POD and the contents of proline, soluble sugar, and soluble protein were all higher in MH than in E under normal conditions. When plants grew under 6 ◦ C stress, the activities of SOD and POD and the contents of proline and soluble sugars increased in both varieties compared with the control (Figure 2A,B,D,E), but the fold increases of soluble sugars were higher in MH (4.8-fold) than in E (2.2-fold) at 6 ◦ C (Figure 2E). After the plants experienced 4 ◦ C stress, the POD activity and the content of soluble sugars all decreased to control levels in E and MH (Figure 2B,E). These results indicate that SOD and POD play an important role in Anthurium’s response to cold stress and that POD activity could be inhibited under a lower temperature; they also indicate that proline and soluble sugars are involved in balancing the osmotic gradient between the cell’s surroundings and the cytoplasm under 6 ◦ C stress. The soluble protein content of E increased while that of MH decreased under cold stress (Figure 2F). This indicats that soluble protein contributs to E’s cold resistance. MH was sensitive to cold even Int. J. Mol. Sci. 2024, 25, 250 20 of 28 though the activities of antioxidant enzymes and the contents of osmolytes were higher than those in E under normal growth conditions. This suggests that it is the quick change in the biochemical indexes and not the starting levels of these indexes that is important in plant cold-resistance. 3.2. Plant Hormone Signal Transduction Pathways Are Important for Cold Response in Anthurium Plant hormones function as vital regulators in the responses of plants to low temperatures [57–59]. Indole-3-acetic acid (a form of auxin), ABA, GA3 (a form of GA), JA, BR, and zeatin (a cytokinin) have been shown to play important roles in enabling plants to cope with stress, and an increase of plant hormones has been found to enhance resistance to cold stress in crop species such as rice [60], tomato [61], cucumber [62], and pear [63]. Common CSR DEGs and DEGs in the turquoise module were found to be enriched in genes involved in the KEGG plant hormone signal transduction pathway, and 226 DEGs were annotated to this pathway. In E, the upstream genes in the auxin and GA metabolic pathways were downregulated while the downstream genes were upregulated. More upregulated DEGs in the BR and JA metabolic pathways, especially DEGs related to JAZ, were found in E than in MH. This indicates that plant hormones and related genes play essential functions in the resistance of Anthurium to cold. The DEGs in this pathway may be good targets for further research. 3.3. Trehalose Metabolism in Anthurium Cold-Response Trehalose is a disaccharide and serves as an energy source, protectant, and signal molecule in plants [11,12]. Trehalose accumulates and trehalose-related genes are induced during plant responses to abiotic and biotic stresses [13,14,64]. Recently, more and more studies have demonstrated that T6P, the intermediate of trehalose biosynthesis, functions as a signal and regulator in balancing sucrose levels in plants [17]. In this study, the upregulated common DEGs in both cultivars under cold stress (both 6 ◦ C and 4 ◦ C) were enriched in trehalose-related terms (GO:0004805, GO:0003825, GO:0070413, and GO:0005992). Analysis of the transcriptome data revealed that 25 DEGs are related to trehalose metabolism and upregulated after cold stress. These results indicat that an increase in trehalose metabolism may be a common plant response to cold stress and are consistent with the results of Mollavali et al. [64]. The DEGs encoding trehalose-related proteins could potentially be targeted to increase plant cold-stress tolerance. 3.4. Ribosomal Proteins Play Important Roles in Cold Tolerance in Anthurium The ribosome is the primary site for protein synthesis, and ribosomal proteins play vital roles in translation, ribosomal structure, and ribosome biogenesis. Ribosomal proteins also play roles in plant salt-stress responses [65,66], and several studies have implicated ribosome proteins involved in cold-stress response [29,31–34]. In this study, the soluble protein content in MH decreased under cold stress while that in E increased, and the greenyellow module identified by WGCNA contained 28 DEGs encoding ribosomal proteins. Seventyeight genes encoding ribosomal proteins were identified as being differentially expressed in response to cold stress in Anthurium (Figure 10A, Table S10). More upregulated DEGs encoding ribosomal proteins were found in E than in MH. The expression patterns of ribosomal protein DEGs were consistent with the changes in soluble protein contents in the two Anthurium varieties. This suggests that the cold-resistant variety enhances protein synthesis in order to cope with the cold conditions, whereas the susceptible one cannot maintain protein synthesis, leading to a decrease in protein content. 3.5. HSP, Pectinesterase, and Plant Defense Proteins Are Commonly Involved in Anthurium Responses to Cold Stress Common DEGs between cold treatment and the control in different comparisons may reflect common cold-response mechanisms in Anthurium. HSPs and HSFs regulate protein folding and have multiple functions in abiotic stress [67,68]. For example, small HSPs Int. J. Mol. Sci. 2024, 25, 250 21 of 28 (sHSPs) exhibit chaperone activity [69], and heterologous expression of a chestnut sHSP enhanced Escherichia coli viability under cold stress [70]. GbHSP16.8 and GbHSP17 were found to be induced by cold stress in Ginkgo biloba [71]. Transgenic Arabidopsis thaliana constitutively expressing PfHSP17.2, as isolated from Primula forrestii leaves, displayed higher resistance to cold compared with wild-type plants [72]. Song et al. (2016) reported that the expression of HsfA3 was induced by oxidative damage in Arabidopsis [73]. HsfAs of bermudagrass were upregulated at 5 ◦ C [68]. In this study, two sHSPs and one HsfA were among the common DEGs upregulated in E and downregulated in MH (Tables 2 and 3), indicating that sHSPs and HsfA may be involved in establishing cold tolerance in Anthurium. The cell wall is a dynamic polysaccharide network that offers the plant stability and protection under cold stress. Pectinesterases, which are ubiquitous cell-wall-associated enzymes, partially determine the mechanical properties and porosity of cell walls by demethylating pectin [74]. These enzymes have been shown to be involved in many developmental processes [75], and because they can strengthen or weaken the cell wall dependent on their mode of action and the environment, they are also likely important in stress response [75]. Consistent with a role for pectinesterases in cold tolerance, genes encoding pectinesterase and pectinesterase-like proteins were upregulated in E and downregulated in MH. Proteins involved in plant defense are also important for plant tolerance to biotic and abiotic stress. Genes encoding members of three protein families involved in plant immunity, SBT, GLR, and CRK, had increased expression in MH and decreased expression in E under cold stress (Tables 2 and 3). SBTs are induced by pathogen infection, are secreted to the plant extracellular matrix, and function in pathogen recognition and immune priming [76,77]. SBTs may be involved in signal recognition in plant immunity. Plant GLRs play important roles in various plant-specific physiological processes, such as pollen tube growth, sexual reproduction, root meristem proliferation, internode cell elongation, stomata aperture regulation, and innate immunity and wound responses [78,79]. GLRs regulate ion fluxes across membranes, particularly the flux of calcium, a key second messenger in plant cell responses to stimuli [78]. GLRs of tomato mediate the cold acclimation-induced chilling tolerance by regulating apoplastic H2 O2 production and redox homeostasis [80]. The increased expression of GLRs may aid ROS scavenging under cold stress. CRKs are a type of receptor-like kinase; they regulate various plant development processes and responses to biotic and abiotic stress [81]. Arabidopsis CRK28 was found to participate in root growth and epidermal cell differentiation and to fine-tune ABA signaling during germination and early root growth [81]. Mou et al. (2021) reported that CaCRK5 was induced in pepper upon Ralstonia solanacearum infection and treatment with SA [82]. CRKs, as kinases, may affect plant hormone signaling and increase cold resistance. These proteins may be common to biotic and abiotic stress responses, and their functions may reflect common mechanisms underlying these responses. The common CSR DEGs are critical for elucidating the molecular mechanisms of Anthurium cold resistance, especially the DEGs upregulated in the cold-resistant cultivar E and downregulated in MH. These may explain the cold resistance of E and should be further studied to understand their function in Anthurium cold resistance. 4. Materials and Methods 4.1. Plant Material and Stress Treatments Two Anthurium cultivars, Elegang (E, cold-tolerant) and Menghuan (MH, cold-sensitive), were used in this study. The full-flowering plants were grown in a growth chamber for 3 days with a 12/12 h day/night cycle at 25/22 ◦ C. For cold treatments, the plants were grown at 15 ◦ C for 3 days, then the temperature was decreased to 6 ◦ C for 3 days and then 4 ◦ C for 3 days. The control plants were grown at 25/22 ◦ C throughout the experiment. Samples were collected at 25/22 ◦ C (control/CK), 3 days after the treatments at 6 ◦ C and 4 ◦ C. Three independent experimental replicates were performed, each consisting of three independent biological replicates. All the samples were immediately frozen in liquid nitrogen and stored at −80 ◦ C for further use. Int. J. Mol. Sci. 2024, 25, 250 22 of 28 4.2. Determination of Enzyme Activity and MDA, Soluble Sugar, and Soluble Protein Contents The collected samples were used for determining the physiological indexes in both cultivars. The activities of superoxide dismutase (SOD, EC 1.15.1.1) and peroxidase (POD, EC 1.11.1.7), and the contents of malondialdehyde (MDA), soluble sugar, and proline were measured according to Cai and Gao (2020) [83]. The content of soluble protein (cat no. BCAP-2-W) was measured using the kits provided by Suzhou Comin Biotechnology Co., Ltd. (Suzhou, Jiangsu, China). 4.3. RNA Extraction and cDNA Library Construction Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s procedure. The amount and purity of RNA in each sample were quantified using a NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed by a Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA) with RIN number >7.0 and confirmed by electrophoresis on a denaturing agarose gel. Poly (A) RNA was purified using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, Waltham, MA, USA) and then fragmented into small pieces using the Magnesium RNA Fragmentation Module (NEB, Cat. e6150, Ipswich, MA, USA). The cDNA was then synthesized using the cleaved RNA fragments as template. After the addition of an ‘A’ tail, size selection of the cDNA was performed with AMPureXP beads. The cDNA library was constructed and subjected to 2 × 150 bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000. 4.4. De Novo Transcriptome Assembly and Analysis Raw transcriptomic data generated by the Illumina Novaseq™ 6000 were preprocessed to remove the reads that contained adaptor contamination, low quality bases, and undetermined bases using the fastp [84] and perl scripts in-house. Then, the sequence quality was verified using fastp including the Q20, Q30, and GC-content of the clean data. All downstream analyses were based on clean data of high quality. De novo assembly of the transcriptome was performed with Trinity 2.4.0 [85]. Trinity groups transcripts into clusters based on shared sequence content, and a transcript cluster is very loosely referred to as a “gene”. The longest transcript in the cluster was chosen as the “gene” sequence (aka unigene). Salmon [86] was used to determine the expression level for unigenes by calculating transcripts per kilobase million (TPM) [87]. The differential expression analysis for two conditions or groups was performed using the R package edgeR [88]. The genes were considered differentially expressed between the treated and the control samples if |log2 (foldchange)| > 2 and FDR (adjust p-value) < 0.001. All assembled unigenes were aligned against the non-redundant (Nr) protein database (http://www.ncbi.nlm.nih.gov/, accessed on 14 April 2021), SwissProt (http://www.expasy.ch/sprot/, accessed on 14 April 2021), Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.kegg.jp/kegg/, accessed on 14 April 2021), and eggNOG (Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups) (http://eggnogdb.embl.de/, accessed on 14 April 2021) databases using DIAMOND [89] with an E-value cut off of 10−5 . Functional classification was performed using Gene Ontology (GO) (http://www.geneontology.org, accessed on 14 April 2021) by DIAMOND [89] with an E-value threshold of 10−5 . The transcriptome sequence data were deposited in NCBI’s Gene Expression Omnibus repository (https: //www.ncbi.nlm.nih.gov/geo, accessed on 21 May 2023), under accession number PRJNA973051. 4.5. Identification of Key Modules Related to Plant Cold Stress by WGCNA WGCNA is a systems biology method for describing the correlation patterns among genes that have similar expression patterns in specific biological functions [47]. In this experiment, a total of 24,695 DEGs were used to construct weighted co-expression modules in the R package WGCNA (version 2.0), using the guidelines from the published tutorials (https://www.omicstudio.cn/analysis/wgcna?id=16, accessed on 14 October 2023). After sample clustering, the soft threshold of module analysis was determined using the scale Int. J. Mol. Sci. 2024, 25, 250 23 of 28 independence and mean connectivity analysis of modules with different power values. The power value ranged from 1 to 30, then the values of scale independence and mean connectivity were calculated accordingly. The power value was calculated when the scale independence value was 0.85. To classify similar gene expression profiles into different gene modules, the average distance with a minimum size threshold of 30 and merge cut height of 0.25 were used to construct a hierarchical clustering dendrogram from the topological overlap matrix (TOM). Moreover, the associations of modules with conditions for the 18 samples were determined using the calculated module eigengenes and Pearson’s correlation coefficient values. The hub genes were selected by the cytohubba plug-in using the MCC algorithm and visualized by Cytoscape (version 3.7.1) software [90]. 4.6. qRT-PCR Analysis The RNA quality was checked by electrophoresis and the concentration was calculated according to the optical density (OD) at 260 nm and 280 nm using a Nanodrop 2000 (NanoDrop, Wilmington, DE, USA). The first-strand cDNA was reverse transcribed from 1 µg of the total RNA using the Evo M-MLVRT Kit (cat no. AG11711, Accurate Biology, Changsha, China). qRT-PCR was performed using the SYBR® Green Premix ProTaq HS qPCR Kit (cat no. AG11718, Accurate Biology, Changsha, China) on the Roche LightCycle96 Real Time System (Roche, Switzerland). The total volume for each reaction was 10 µL, comprising 0.4 µL specific primers, 1 µL cDNA, 5 µL SYBR mixture, and 3.6 µL double-distilled H2 O. The cycling program was as follows: 10 min at 95 ◦ C, followed by denaturation at 95 ◦ C for 15 s, annealing, and elongation at 60 ◦ C for 30 s (40 cycles). Gene-specific primers were designed by Primer Premier 5.0 software (Table S11). The actin gene was used as an internal reference gene. The qRT-PCR expression analysis for each gene was performed using three technical replicates (with three biological replicates). The relative expression levels of genes were calculated using the 2−∆∆CT method [91]. The expression level of CK for E was used as a control; its value was set to 1. The relative expression levels of other samples (including the CK for MH) were determined relative to the control. In this way, we can compare the same gene across all samples of the two cultivars. 4.7. Statistical Analysis The statistical analysis was performed using GraphPad Prism 8.0. The statistical significance was determined by a multiple t-test. Error bars represent SD (n = 3). 5. Conclusions The main purpose of this study was to investigate the molecular mechanisms underlying the cold response in two Anthurium cultivars with contrasting cold tolerances, through analysis of morphological and physiological changes combined with analysis of the transcriptome data. Soluble protein content was identified as being vital for cold resistance. HSPs and HsfA, cell-wall-associated pectinesterases, and plant defense proteins are common components of cold-stress responses in both cultivars but have opposite expressions patterns. They may play important roles in Anthurium cold resistance and can represent targets for enhancing Anthurium cold tolerance. Finally, plant hormone signal transduction pathways, trehalose metabolism, and ribosomal proteins are important for the cold resistance of Anthurium. Our results suggest that proteins involved in protein synthesis (ribosomal proteins, HSPs, and HsfA), energy metabolism, and signal transduction pathways play essential roles in Anthurium cold resistance and should be further studied. Supplementary Materials: The supporting information can be downloaded at https://www.mdpi. com/article/10.3390/ijms25010250/s1. Figure S1: GO categories of 132,018 unigenes. Figure S2: KEGG category of 132,108 unigenes. Figure S3: Number of all DEGs. (A) DEGs from comparison of cultivars. (B) DEGs from comparison of cold treatment vs. control. Figure S4: KEGG analysis of cultivar-only DEGs in common 9132 DEGs. Figure S5: GO analysis of common DEGS for the same treatment between two cultivars. Figure S6: Scatter plots for KEGG pathway enrichment analysis of DEGs in the four key modules. (A) KEGG analysis of DEGs in the turquoise module; (B) Int. J. Mol. Sci. 2024, 25, 250 24 of 28 KEGG analysis of DEGs in the greenyellow module; (C) KEGG analysis of DEGs in the tan module; (D) KEGG analysis of DEGs in the yellow module. Only the top 20 KEGG pathways are shown in the plots. Figure S7: Relative gene expression analysis of 16 randomly selected DEGs by qRT-PCR. Comparison of qRT-PCR with fold-change values calculated using transcriptome data from E and MH cultivars. The relative expression level is presented on the left y-axis, and the fold-change values from the transcriptome data are shown on the right y-axis. The relative expression levels of 16 common DEGs were investigated using qRT-PCR. The results from three replicates were analyzed using the 2−∆∆Ct method and normalized to the actin housekeeping gene. The data shown are the means of three biological replicates (n = 3), and the bar represents the standard deviation (SD) for each mean value. Table S1: Overview of the RNA sequencing reads for the 18 A. andraeanum leaf samples. Table S2: All enriched GO terms for the 132,018 unigenes. Table S3: DEGs between cold-tolerant and cold-susceptible cultivars under the same temperature conditions. Table S4: A list of cold-responsive genes in E and MH. Table S5: All information about the 9132 common DEGs. Table S6: The enriched GO terms for the four key modules. Table S7: The top 20 hub genes of the four key modules. Table S8: The log2(fold-change) of DEGs involved in plant hormone signal transduction pathways. Table S9: The 25 trehalose-related DEGs. Table S10: The 78 DEGs encoding ribosomal proteins. Table S11: Primer sequences for qRT-PCR analysis of DEGs. Author Contributions: Conceptualization, C.W., Y.F. and S.F.; Methodology, N.D. and L.L.; Software, C.W.; Validation, N.D.; Formal Analysis, N.D. and L.L.; Investigation, N.D.; Resources, L.L.; Data Curation, C.W.; Writing—Original Draft Preparation, C.W. and N.D.; Writing—Review & Editing, C.W., Y.F. and S.F.; Visualization, C.W.; Supervision, S.F.; Project Administration, C.W.; Funding Acquisition, C.W. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Innovation Project of Top Ten Agricultural Characteristic Industrial Science and Technology of Ji’nan and Shandong Province Natural Science Foundation (ZR2018MC001). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. 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Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 Different approaches to determining the employer brand were analyzed. The main components of the employer brand were identified. It is customary to divide the components of the employer brand into tangible and intangible ones. Material ones include wages, official employment, the material system of motivation. Intangible components are additional benefits, office, rating, and reputation of a company, type of activity of a company, the possibility of training and development. A tendency of the lack of information to form the employer brand for Generation Z was detected. Generation Z is the people born in 1997–2012. The list of value propositions of employers in the labor market for Generation Z was analyzed. The results of our own research – a survey among students (1341 respondents) having or not having experience – were analyzed. To analyze the results of the survey, mathematical expectation, root mean square deviation, and coefficient of variation of expert evaluation results were used. The rating of the components of the employer brand and their integrated scores in terms of attractiveness for Generation Z were established. The aspects to which employers need primarily to pay attention to for forming a value proposition in the labor market when selecting personnel were established This study is important because the main components of the employer brand and their priority for Generation Z were not investigated in the scientific field. As a result of the study, companies were rated by their attractiveness as a place of work or internship in retail and FMCG (fast-moving consumer goods) spheres. These data can be used by HR-brand agencies, HR departments of international and national companies. Using the results of the survey of the formation of the employer brand for Generation Z will allow enterprises to take into account the expectations of their target audience Keywords: employment of students, labor market, labor distribution, internal brand, organization of HR management, employer brand 1/13 ( 115 ) 2022 UDK 330 DOI: 10.15587/1729-4061.2022.252549 ANALYSIS OF EMPLOYER BRAND FOR YOUNG PEOPLE Maryna Dzhulai Corresponding author Postgraduate Student* E-mail: maryna.dzhulai@gmail.com Iryna Fedulova Doctor of Economic Sciences, Professor* Iryna Bolotina PhD, Associate Professor Department of Labour Economics and Management National University of Food Technologies Volodymyrska str., 68, Kyiv, Ukraine, 01601 *Department of Management Kyiv National University of Trade and Economics Kyoto str., 19, Kyiv, Ukraine, 02156 Received date 30.11.2021 How to Cite: Dzhulai, M., Fedulova, I., Bolotina, I. (2022). Analysis of employer brand for Accepted date 17.01.2022 young people. Eastern-European Journal of Enterprise Technologies, 1 (13 (115)), 80–91. doi: https://doi.org/10.15587/1729-4061.2022.252549 Published date 28.02.2022 1. Introduction The formation of the employer brand is an urgent issue for company positioning in the labor market, creating its reputation, and maintaining its image. This is due to the importance for each company to provide itself with the staff of the necessary intellectual and emotional level that is ready to develop along with the company development. The COVID-19 pandemic has affected the employer market and created new approaches to the work of employees. Companies are gradually recovering from the crisis due to a change in the business model, job cuts, and the creation of new products and services. In addition, human resource managers face a new challenge – to motivate and organize remote work of employees. In 2020, 8.8 % of employees worldwide lost their jobs. As a result, the employees in the region of Europe and Central Asia suffered from redundancy most of all. The International Labor Organization predicts that unemployment will be reduced to 75 million in 2021 and to 23 million in 2022. In the scientific sphere, new tasks appear: to investigate the influence of external circumstances on the formation of the employer brand during remote work of employees, to identify new components of the brand, to analyze channels of communication with the target audience of the brand. As a result, the formation of the employer brand is very important and requires well-grounded management decisions. First of all, remote work influenced its components and the format of reporting to employees and potential candidates. Secondly, employees choose a company to work for, looking at intangible components of motivation during remote work. That is why companies have the challenge to differ from other companies in the labor market, help the employees who work from home to feel the values, mission, and spirit of the company. The value proposition of the employer brand should also include components for the employee’s family. The factor of communication with employees during a crisis is becoming important. The “small talk” format in the chat with colleagues or during a video call is a result of the transformation of the “small talk” format over coffee in the office. A new trend in companies is the preservation of the mental and physical health of employees. Of course, most companies are primarily concerned about physical health. However, employees have a new challenge – to improve the skills of time management, because the usual 9 to 18 framework of the working day is disappearing. Formation of the employer brand for students, in our case it is mostly Generation Z, needs more research. It is necessary to take into account the specific characteristics of the behavior of Generation Z in the process of creating a working environment during remote work and choosing components of the employer brand for young people. Generation Z has more advanced digital skills and starts working earlier compared to Generation Y. However, remote work helps students to combine work and study. According to the research by Dell Technologies in 2020, 20 % of employees are representatives of Generation Z. That is why there is a need to identify the main components of the value proposition of an employer for students when they are looking for a would-be job. That is why scientific research into the formation of an employer brand for Generation Z under the influence of COVID-19 is relevant. 80 Copyright © 2022, Authors. This is an open access article under the Creative Commons CC BY license Transfer of technologies: industry, energy, nanotechnology 2. Literature review and problem statement Paper [1] states the need to attract candidates with the necessary set of skills and knowledge and create all conditions for each employee to feel the brand and mission of the company, as well as to ensure the implementation of the set goals. But for modern companies that hire representatives of Generation Z, knowledge, skills and understanding of the goals and mission of the company is an important, but not a decisive factor. For them, creativity, information literacy, emotional intelligence are the most desirable parameters for potential job candidates. Research [2] showed that branding includes two areas: marketing and management of the staff who is involved in work. This indicates two dimensions of the formation of the employer brand: regarding its creation and management in the activities of an enterprise. The employer brand and the company’s brand, despite having many features in common, still differ significantly. To form the employer brand for Generation Z, the priority is not marketing, but rather part of personnel management that is responsible for its development and determines the possibility of building a career. The authors of [3] believe that the employer brand should be considered more broadly since the strategy of forming the employer brand is the first strategy. The strategy helps to manage human resources, ensures the competitiveness of an enterprise in the market, and allows remaining successful leaders. However, there is a need to determine the causes and prerequisites for the influence of the employer brand on the financial results of a company. According to [4], the employer brand includes the process where a company and its employees sign an agreement according to which a potential employee has a reason to become part of the company’s team and a company has a reason to hire an applicant. This approach is very simplified, it is the identification of these reasons that is necessary to form effective value propositions. The main characteristics of the employer brand are given in [4], in particular: economic, including compensation and benefits, functional (when an employee receives a certain skill), psychological (self-identification, comparison with the status of a company). Such division of the characteristics of the employer brand may be appropriate, but the list of economic, functional, and psychological characteristics of the brand requires clarification. Paper [5] states that the employer brand is a set of qualities of a company, through which it is associated with the target audience. It includes unique and positive qualities of tangible and intangible advantages of working in a company. However, it is necessary to study what qualities the employer brand should be characterized by for young people in Generation Z. An important scientific characteristic is presented in paper [6], where the employer brand is considered as an emotional and stable image of a company in the labor market. A similar scientific confirmation of the characteristics of the employer brand is given in paper [7]. It is defined as a set of economic, professional, psychological benefits that candidates receive while working in a company. This is a set of basic values that distinguish the company in the labor market. According to [8], the employer brand is also considered as a company’s reputation and image. In this case, the goal of a company is to find and attract qualified candidates for vacancies. However, the image of a company and its reputation in the labor market are created during communication between a company and existing and potential employees. Accordingly, the employer brand should include unique characteristics that make it com- petitive and attractive in the labor market. There is a need to search for these unique characteristics. An additional feature of the employer brand is found in research [9]. In it, the brand is characterized by two levels of the employer brand. Firstly, it is the result of the functioning of the strategy or methods for developing the employer brand and the employee’s brand. Secondly, it is a complex of the company’s HR brand, which includes corporate culture, communication between internal management departments, and external market participants. Probably, consideration of three levels of the employer brand is more exact: the employee’s brand, the company’s HR brand, the company’s corporate brand. It is this view that will be most useful in the formation of value propositions of the employer for Generation Z. The employer brand is defined as the management influence of a company in order to communicate with potential and current employees in terms of creating the most attractive place of work. The brand includes the construction of a corporate brand as the company image and a comfortable workplace for the development as a professional. A similar proof was found in research [10], where the employer brand is defined as internal measures with effective feedback with employees, tangible and intangible motivation, an effective system for assessing personnel, the possibility of developing and training employees. It is important that this work considers one of the highest priority value propositions for Generation Z. This is the possibility of training and development of employees. However, this list is incomplete and needs to be clarified and supplemented. Paper [11] proves that HR-brand management is a trend that affects not only the company from the inside but also determines the position of an enterprise in the labor market. A good reputation of the employer brand affects the effectiveness of HR processes, namely, it creates an opportunity to attract new talents, retains the company’s staff, and allows enhancing employee motivation by 0.5 percentage points and increases profits by 0.8 percentage points. However, to determine the relationship between the reputation of the employer brand and the company’s effectiveness, it is necessary to predetermine the level of development (maturity) of the employer brand. To do this, it is necessary to analyze the characteristics of its formation and, first of all, value propositions. According to the study reported in paper [12], the corporate brand is considered an integral part of the employer brand. At the same time, the most important factors of its formation are the labor market, communication between a company and applicants, value propositions. It is most accurate to consider the employer brand as part of the company’s corporate brand, and not vice versa. The list of factors for the formation of the employer brand considered in this paper is incomplete and needs to be supplemented. A high level of the employer brand simplifies the measurement of brand capital and associativeness for job seekers [13]. The lower the level of the employer brand, the more complex and complicated the business processes of the HR department become for analysis and evaluation. Such a conclusion is appropriate, but to prove this provision, it would also be useful to formulate methodological provisions to assess the level of the employer brand. A new direction in the field of brand management is Emotional Text Mining [14]. This means extracting brief information from a large amount of text in quick mode to understand the essence of the process. In brand management, by analogy, this can be considered as an analysis of a company as an em- 81 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 ployer in social networks. This approach is especially important for Generation Z. When searching for information about the employer, they are guided to a greater extent by social networks and a small amount of information messages. Companies should take this into account, so there is a need to minimize the number of value propositions while making them more meaningful. According to the empirical study [15] in Germany in 2020, it was noted that the higher the level of conformity of external communication with job seekers with the actual components of the employer value proposition, the better the perception of the brand. A realistic promise to job seekers increases their loyalty and trust after being employed in a company. During the development of the brand strategy, management must maintain a high level of brand recognition and uniqueness and consistency of its formal components [16]. However, the actual components of the employer value propositions and types of external communications with job seekers need clarification. Paper [16] considers new requirements for personnel, which are put forward by the labor market today under conditions of the Fourth Industrial Revolution. After all, the changes that occur in the context of digitalization of the economy in supply and demand in the labor market change the requirements of employers for vacancies and requirements of young people for a potential place of work. But specific skills and knowledge related to the digitalization of economic processes are not fully defined, both in terms of employer requirements and in terms of the requirements of potential job seekers. The study [17] showed the importance of ensuring the compliance of the structure of training young people with the current and prospective needs of the labor market, and the requirements of employers. This paper also deals with the trends in the development of competencies, which are a prerequisite for determining the value propositions of employers in the labor market. That is why there is a need to critically comprehend modern trends in the development of the labor market and form a list of value propositions of an employer that meet them. According to study [18], the dynamics of changes in supply and demand in the labor market are determined not only by the current trends of the fourth industrial revolution but also by the COVID pandemic. Thus, in the labor market today there is a transformation of the behavior of business entities at the production, consumption and management level. Thus, quarantine restrictions today require consideration and reflection in the workplace requirements of a would-be job applicant and the employer’s capabilities to meet these requirements. The employer brand as a technique for influencing the maintenance of existing employees in an IT company shows positive results [19]. In the presence of a high-quality brand, there appear brand supporters who spread information on their own. The cyclical nature of branding is manifested in the fact that, on the one hand, it acts as a source for the company’s reputation in the labor market, and on the other hand, the business rating and reputation of a company should be included in the list of value propositions of the employer brand. In the above-mentioned scientific sources [1–20], separate value propositions of employers were considered to a certain extent. However, the characteristics that affect the formation of the employer brand for Generation Z were not fully explored. There remains an unresolved problem of identifying and systematizing the most complete list of employer value propositions for Generation Z, which takes into account current labor market trends, digitalization of the economy, pandemic challenges, employers’ requirements for the staff, and requirement of potential job seekers for the desired workplace. 82 1/13 ( 115 ) 2022 3. The aim and objectives of the study The aim of this study is to identify factors and components of the employer brand for Generation Z. Generation Z makes up most of the candidates for initial positions in a company. This will help employers to form an employer brand for young people, increase the number of candidates for one position, provide themselves with employees of the necessary intellectual and emotional level. To achieve the goal, the following tasks were set: – to analyze the rating of employer attractiveness in the labor market for Generation Z according to the results of an expert survey; – to determine the list of value propositions to form the employer brand for Generation Z, to conduct an integrated assessment of their level of significance according to the results of an expert survey; – to analyze the job format attractiveness for Generation Z according to the results of an expert survey. 4. The study materials and methods The object of this research is the employer brand. The subject of the study is the theoretical and practical provisions of the formation of the employer brand. The main hypothesis of the study is that in order to form, analyze and determine the level of the employer brand for Generation Z, it is necessary to form a list of brand value propositions and that determining the integral estimate of their significance should solve the problem of employers’ formation of information messages, communication measures and value propositions for Generation Z. Hypothetical assumption 1 is that when Generation Z chooses an internship place and decides whether to stay in a company after the internship, value propositions that are related to training and development and directly affect career formation will be the top priority. Hypothetical assumption 2 is that for Generation Z, even after the end of the quarantine, the most attractive will be the mixed format of work, in which part of the work takes place in the office, and another part at home. The monographic method and the method of theoretical generalization were used to determine scientific approaches to understanding the essence of the employer brand. The systemic approach was used to systematize the value propositions of an enterprise in the labor market. The method of expert evaluation was used to determine the priorities of value propositions of employment, internship, and choice of form of work during the quarantine. Graphic methods were used to illustrate theoretical and practical materials. Statistical analysis was used to analyze the results of the expert survey. According to the results of the literature review [2, 8, 9, 11, 12, 21–24] and job sites, we generated a list of value propositions, main features, and characteristics of enterprises based on which they form the employer brand and create attractiveness in the market for a job candidate. The survey of the student audience was conducted in May 2021 in cooperation with Ukrainian universities and student organizations. The survey included 1,341 respondents who prioritized a company in each industry for internship and work. The audience of respondents included: 776 students without work experience and 565 students or graduates with experience from 1 to 5 years. Transfer of technologies: industry, energy, nanotechnology To determine the TOP companies that offer internships and vacancies for students and would take part in the study, the career sites, preliminary brand ratings of employers of TOP companies in each area, and their market share were analyzed. The TOP-9 retail companies, TOP-12 FMCG companies, and TOP-13 IT companies were selected for the study. The choice of directions of companies’ activity was determined by the fact that today’s students chose high-tech companies as a workplace. The criteria of selecting a company for internships were also formed, which made it possible to compare the choice of companies by students and graduates with and without experience, to determine a convenient work schedule for students after the COVID-19 pandemic. The expert survey was conducted online using Google Form. To analyze the results of the expert survey, Microsoft Excel software (USA) was used. During analysis, the Pareto diagram was used, which helped to identify and visually separate 20 % of value propositions that affect 80 % of the results of employer brand formation. Using the graphic evaluation, we determined the criteria that will allow HR managers to manage the company’s value propositions during the formation of the employer brand and develop a set of measures for their implementation in the activities of companies. Mathematical processing of survey results was carried out based on the probabilism approach. In accordance with this, mathematical expectation, standard deviation, and factor of variation of the resulting survey scores were determined. Indicators of mathematical expectation, standard deviation, and variation factor were used to summarize the survey results and determine the generalized level of significance and priority of value propositions to choose the place of internship and work by Generation Z representatives. 5. Results of the study of employer brand formation 5. 1. Analysis of the rating of employer attractiveness in the labor market for Generation Z The employer brand should be considered as a totality of internal and external factors. Firstly, it is the sum of qualities, characteristics and emotions that arise in employees and potential candidates. Secondly, it is the sum of tangible and intangible factors that form a positive image for the target audience. Thirdly, the structure of the employer brand is formed for an internal audience (employees) and external (candidates). The employer brand for students has a unique set of characteristics. This is caused by the specifics of Generation Z. Generalization of research results made it possible to determine the main components of the employer brand for Generation Z. To determine their importance and ranking, a survey of students was conducted. The survey audience is students of Ukraine without work experience or with experience up to 1 year studying in the 3rd year of bachelor studies (23.7 %) (Fig. 1). In addition, the survey audience was studied according to the areas of study: business, law, economics, management, and sociology (Fig. 2). Year of studying 3 year of baccalaureate 2 year of baccalaureate 1 year of baccalaureate 4 year of baccalaureate 1 year of magistracy Graduate (1 year after graduation) 2 year of magistracy 0,60% 0,00% 16,60% 7,20% 5,50% 5,00% 10,00% 15,00% 23,70% 22,90% 20,50% 20,00% 25,00% Fig. 1. The share of respondents according to the year of study at university Social sciences (business, law, economics, management, sociology, etc.) 7,20% Technical sciences (engineering, metallurgy, machine building, etc.) 11,10% Exact sciences (cybernetics, informatics, mathematics, etc.) 12,90% 60,10% 8,80% Humanities (history, philosophy, etc.) Natural sciences (geography, physics, chemistry, biology, medicine, etc.) Fig. 2. The share of respondents in accordance with the major The share of re ondents in accordance with the majo Among retail compa of respondents chose popularity of the compa s is company ( e ships of students, explain and. Ro 83 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 Among retail companies, Rozetka is the priority for internships of students, 65 % of respondents chose this company (Fig. 3). This is explained more by the level of popularity of the company’s trading brand than the employer brand. Rozetka is an e-commerce company that sells various goods from food staff to electronic devices. Fozzy Group, which has offline grocery stores throughout Ukraine, ranks second in the rating. The least attractive company for internships is ATB, which does not want to attract students to work. According to analysis, ATB is a more attractive workplace for candidates over the age of 40. Among the FMCG companies, Nestle company (63.1 %) is the most attractive for students without work experience (Fig. 4). Coca-Cola HBC Ukraine ranks second. The least attractive company is Ab InBev Efes. However, companies “Corona”, “Bud”, “Staropramen” are popular and recognizable brands for students. 1/13 ( 115 ) 2022 Most of the companies mentioned in the study are international. Generation Z includes young people born in 1995–2012. This allows using the research results internationally in the scientific field to form the employer brand among young people. In addition, the company Deloitte conducted a survey of 1,531 representatives of Generation Z in 2020 in the United States [15]. It was found that the peculiarities of the character, outlook, and the way of thinking of Generation Z should be taken into account by the company. First of all, 77 % of respondents are active public figures. Young people appreciate volunteer and social projects in the company and pay special attention to it. Secondly, a new trend in the labor market is the choice of work in a startup or a young company, rather than in international companies. A particularly interesting area of work is the technical field (51 %). This entails additional efforts for companies from other fields of recruitment. 70,00% 65% 60,00% 50,00% 40% 41,10% 39,47% 40,00% 27,90% 30,00% 20,00% 10,00% 12,50% 30,40% 31,90% 16,70% 17,88% 15,04% 4,78% 32,80% 20,00% 17,70% 22,48% 27,61% 7,43% 0,00% Student having no experience or up to 1-year 1 year experience Student or graduate having 1‒5 years of experience Top retail c mpaniesZ for companies with Fig. 3. Top retail companies for Generation withGeneration or without Z work experience, % of respondents of responden 70,00% 60,00% 50,00% 40,00% 30,00% 20,00% 63,10% panies, ) is he most attractive 60,50% per Cola HBC U aine 43,19% y is Ab InBev Efes. However, c panies pular and recognizable brands for stu ents 47% 47,10% 45,49% 43,19% 40,20% 38,05% 31,86% 25,84% 25,50% 29% 33,30% 33,30% 35,10% 34,16% 32,74% 30,80% 26,73% 25,31% 22,12% 21,42% 19,12% 16% 10,20% FMCG co for students without work e The least attractive comp are p 10,00% 0,00% Student having no experience or up to 11-year experience Student or graduate having 1‒5 years of experience Fig. 4. TOP companies in FMCG for Generation Z with and without work experience, % of respondents 84 compani of responden in eneration Z with a Most of the companies companie mentioned in the study are international. internat nal. Generation Z Transfer of technologies: industry, energy, nanotechnology All this allows arguing that the influence of a generation on the formation of the employer brand and brand components can be applied internationally. This is due to similar problems and behavior of modern youth. (29.91 %), internship schedule (39.05 %), additional benefits (21.11 %), and the status of an international company (16.86 %) are important but not the major factors. The least important factors are the possibility of relocation to another city /country (16.75 %) and the level of the company’s scale (12.10 %). Fig. 5 shows the results of an expert survey on determining the priority of value propositions of the employer brand for students with or without work experience when choosing a company for internship. To summarize the results of assessing the level of significance of factors for choosing a company for an internship, we used the probabilistic approach, which is based on determining mathematical expectation, root mean square deviation, and coefficient of variation of the survey rating results. 5. 2. Determining, analysis, and integrated evaluation of the significance of value propositions for choosing an internship place for Generation Z Processing the survey results showed that the most important factors for choosing an internship place for students are the opportunity of training at a company (56.96 %), the salary level (44.79 %), official employment (40.85 %). These factors must be included in the vacancy description and promo text for recruitment for internship programs. The company’s business rating and reputation Rank of the sinificance level of the factor at choosing a company for internship Training at company 8 Salary level 17 Business rating and reputation of a company 186 53 168 Working in international company 72 165 Active social position of CEO 91 164 Additional benefits (insurance, working place, etc.) 68 130 Possibility of relocation to another country 151 94 Scale level of a company (number of countries where a company is represented) 131 0 298 195 274 219 89 Office location 360 220 193 65 Positive impact of goods and services on the environment 3 ‒ important 227 159 31 Balance between "good actions" and profit 303 320 119 34 348 317 232 162 65 Internship schedule 4 ‒ very important 308 104 Official employment 442 258 68 305 232 309 228 301 219 310 235 243 252 274 278 50 100 150 200 250 300 350 400 450 500 2 – least important 1 ‒ unimportant Fig. 5. Rating the level of importance of value propositions for students with or without work experience when choosing a company for internship, number of respondents 85 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 The significance of factors was assessed by the following categories: unimportant (grade 1); least important (grade 2); important (grade 3); very important (grade 4). Thus, a scale of estimates of the significance level from 1 to 4 was used. At stage 1, the probability of obtaining an appropriate estimate for each factor is determined from the formula: Х ij = aij ∑ 4 a , (1) j =1 ij where Xij is the probability of obtaining score j for the і-th factor; aij is the number of students who gave score j to factor і; і is the factor index (і=1, …, n); n is the number of factors; j is the value of the factor score ( j=1, … 4). As a result, we get a matrix of probabilities of scores of answers to questionnaire questions: X11  X1 X12  X2 X13  X3 X14 . X 4 (2) Each line of this matrix is a grouped statistical series of probabilities of obtaining the j-th score for each i-th studied factor of the system of assessment of choosing a company for internship. Thus, for a generalized assessment of the factor, it is possible to choose indicators of mathematical expectation, root mean square deviation, and variation factor. The mathematical expectation of the score for each i-th factor can be calculated from the formula: 4 М ( Х i ) = ∑ j ⋅ X ij . (3) j =1 The mathematical expectation of the factor scores shows the average score of the factor according to a fourpoint scale. Root mean square deviation for each i-th factor is calculated from the formula: σ ( Х ) = ∑ ( j − М ( Х )) . 2 4 j =1 i (4) Root mean square deviation shows the magnitude of fluctuations in the score of each factor around its mathematical expectation. Variation factor is calculated from the formula: CV ( X i ) = σ ( Х ) М ( Хi ) ⋅100 %. (5) Variation factor shows how many fluctuations in percentage fall per unit of the expected score of the factors. With the help of this indicator, it is possible to determine how large the differences in the assessment of this factor by students are. The results of statistical analysis of the survey results are shown in Table 1. The factor of variation of the survey results showed the magnitude of differences in the scores of respondents for each factor under study. The least similar among respondents is the opinion about the possibility of relocation to other countries. This factor has the greatest divergence in 86 1/13 ( 115 ) 2022 the respondents’ opinions. The most similar is the assessment of the possibility of training in a company (20.13 %). According to the generalized assessment of the level of significance of the factor, it is possible to construct a Pareto diagram (Fig. 6). With the help of such analysis, it is possible to identify the most significant factors for determining the place of internship and work, which account for 80 % of students’ choices to substantiate the place of internship and work. That is, these factors determine 80 % of the company’s attractiveness for students when choosing an internship place. Table 1 Integrated score of the significance of value propositions at choosing an internship place by representatives of Generation Z Value propositions Level of company’s scale (for example, the number of countries, where a company is represented) Possibility of relocation to another city/country Additional benefits (insurance, working place, etc.) Active social position of СЕО Working in an international company Office location Positive impact of good/services on the environment Balance of “good actions” and profit Business rating and reputation of a company Internship schedule Official employment Salary level Training in company Probability of obtained M(Xj) Ơ(Xj) CV (Xj) scores 0.17 0.36 0.35 0.12 2.43 0.91 37.41 0.19 0.32 0.31 0.17 2.45 0.99 40.18 0.09 0.30 0.40 0.21 2.73 0.89 32.57 0.12 0.28 0.39 0.21 2.70 0.93 34.63 0.09 0.29 0.40 0.22 2.74 0.90 32.91 0.07 0.30 0.39 0.24 2.80 0.88 31.36 0.11 0.28 0.35 0.25 2.74 0.96 35.07 0.08 0.25 0.38 0.28 2.87 0.92 32.15 0.04 0.20 0.46 0.29 3.01 0.81 26.94 0.04 0.08 0.02 0.01 0.83 0.98 0.77 0.70 26.50 32.17 23.61 20.13 0.15 0.21 0.13 0.09 0.41 0.30 0.40 0.33 0.39 0.41 0.45 0.57 3.15 3.03 3.27 3.46 Analyzing the Pareto diagram (Fig. 6), we can say that working in an international company creates additional benefits for students in the form of the possibility of relocation to another country/city and the scale level. These factors do not require additional attention of management but can be included. It is possible to rank factors by the degree of importance using the Sturges method of analytical grouping, according to which the grouping interval is determined from the formula: I М (Х ) = М ( Х )max − М ( Х )min 1 + 3,322 ⋅ lg n , (6) М(Х) max and М(Х) min are, respectively, maximum and minimum values of mathematical expectation of significance of factors; n is the number of unit of totality. Transfer of According to the generalized ge ralized assessment of the level of significance ignificance of the it is possible to co truct 6). Wi t entify the most significant factors r determining the place of internship and rk, which account for 80 of s dents technologies: industry, theenergy, place nanotechnology of internship int nship and work. That is, these factors factor determi attractivene for students when choosing an internship attractiveness inter hip place. Pareto diagram for alysis of value s for ch sing an internship Fig. 6. Pareto diagram forrepresentatives analysis of valueofpropositions choosing an internship place by representatives of Generation Z place by Generation enerationfor Z (by their level of significance) signific nce) (by their level of significance) zing the Paret diagram ( 6), we can say t t work Table 2 shows the identification of the integrated score nificant aspects of management during the employer brand international company creates cre es additional benefits for students in the form of the of the significance of the factors by which students choose formation. to nother country/city and the lev . the internship place and determines the factors that were Using (1) to (5), the importance of factors for students attent n but can be include . included in these categories of assessment. after the internship to continue working in the company was Categorizing the level of importance of the factors in assessed. Fig. 7 shows the ranking of actors that influenced students’ choice of an internship place allows the company the decision to stay in the company after the internship for to focus its efforts and resources on solving the most sigGeneration Z according to an expert survey. Table 2 Grouping the values of integrated score of the significance of the factors when choosing the internship place by Generation Z representatives Integrated score of factors’ significances Category of factors’ significance Qualitative characteristic Factors included in this range ]2,43; 2,63] Low The level of impact of factors is low, they meet the requirements of only a small number of students and are the least productive to create the attractiveness of the employer brand Possibility of relocation to another city/country, business rating of a company ]2,63; 2,83] Below average Factors are not able to actively adapt to most of the requests of students regarding the choice of internship place, their impact on the company attractiveness is not decisive Office location, work in an international company, active social position of CEO, additional benefits ]2,83; 3,03] Average The level of impact of the factors is average, they meet the conditions of the requests of some students, some of them are on the boundary of making decisions on the choice of an internship place Balance between good actions and profit ]3,03; 3,23] Above average The level of impact of factors is high, they are attractive enough for most students when choosing an internship place Official employment, internship schedule ]3,23; 3,47] High Factors have the most productive impact on students’ choice of internship place and create attractiveness of the employer brand in the market Salary level, training in company 87 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 1/13 ( 115 ) 2022 Ranking of factors that influenced the decision to stay for working in a company after internship Possibility to combine work and studies Team Access to training in a company Payment of internship Work in different departments 305 358 274 335 89 149 43 77 Project importance for results of a company 37 0 4 ‒ very important 107 18 International experience 326353 307338 87 24 Adaptation program offered by a company 396 258 101 24 Set goals 418 295 10 Mentor support 460 251 21 Communication with CEO 475 254 12 53 12 51 Interesting project 502 190 3549 1334 100 3 ‒ important 190 204 211 204 186 200 250 293 285 349 300 400 2 – least important 500 600 1 ‒ unimportant Fig. 7. The results of assessment of the factors influencing the decision to stay for working in a company after internship for representatives of generation Z, the number of respondents The most important factors influencing the decision to stay after an internship at the company are the ability to combine work and study at university (average score 3.59 out of 4), team (3.54), and access to training at the company (3.54) (Fig. 7). Score 4 is the highest. The factors that need to be included in the internship program are a flexible schedule and the possibility to work remotely, the presence of a mentor for the internship period, the opportunity to study at the company and develop as a professional (Fig. 7). The tasks set for the internship period, the importance of a project for a company, and the adaptation program in a company are important but not priorities. The least important factors are work in different departments and international experience. 5. 3. Analysis of the attractiveness of the work format for generation Z After the pandemic, a convenient work schedule for students is three working days in the office and two working days remotely (31.3 %) (Fig. 8). The remote format of work combined with meetings with the team in the office ranks second (22.7 %). The hybrid format is ideal. Employees can choose their own work schedule. It should be added that only the remote format of work is unacceptable for students (13.4 %). 63.4 % of Generation Z plan to work in the specialty (63.4 %). Accordingly, this is an ideal option for a company, since the process of finding candidates for internships will be easier for the HR team. Only 5.7 % of respondents do not plan to work in the specialty. What work format is convenient for us? (taking into account the fact that quarantine will be over) 3 working days in the office, 2 working days remotely 31,30% Remote work combined with meeting the team in the office Work in the office for 5 working days 22,70% 16% 4 working days in the office, 1 working day remotely 13,40% Remote work for 5 working days 13,40% Fig. 8. The share of surveyed representatives of Generation Z regarding convenient work format 88 Transfer of technologies: industry, energy, nanotechnology 6. Discussion of the results of the research into employer brand formation for Generation Z The study of the priority place of work for representatives of Generation Z was conducted to identify the best practices for the formation of value propositions by modern companies that willingly hire young people. Indirectly, this determines the companies with effective employer brands. It was found that the most desirable place of work for Generation Z in the retail sector is Rozetka (Fig. 3), which is caused by the popularity of the trade brand, the level of informatization of business processes in the company, flexible work schedule. In the FMCG area, company Nestle, which is an innovative company that values creativity of thinking and can give experience under international business rules, turned out to be the best company for a potential job (Fig. 4). The least attractive workplaces are ATB and Ab InBev Efes (Fig. 3, 4, respectively). Unlike [5], which indicates only attractive areas for generation Z, the results of the study revealed exemplary companies with an established employer brand. This allows additional analysis of the most attractive employers’ best practices regarding the formed value propositions. The review of literary sources [1, 2, 4–7, 11–13, 16–21] allowed constructing a list of value propositions of employers for Generation Z. Mandatory components are training in a company, salary level, official employment, internship schedule, additional benefits, business rating and reputation of a company. Secondary components include the balance between “good actions” and profit, the positive impact of goods/services on the environment, the location of the company, work in an international company, an active social position, the possible relocation, the scale level. A high level of the employer brand in HR management is defined as the formation of a value proposition for employees and job seekers with unique qualities and characteristics for a company in the labor market. A mandatory characteristic of the employer brand is the attraction of talents and the creation of a competitive enterprise [9, 15]. It is considered from two positions: marketing and management, that is, it has a focus on two target audiences [7, 13, 16]. Also, the employer brand has an emotional connotation and creates a certain image for job seekers, which is associated only with a certain company [19, 20]. It was found that the effective employer brand has a positive effect on the company’s performance [14, 21]. The results of the quantitative analysis of the survey on the attractiveness of value propositions made it possible to identify their specific features for Generation Z. Analysis of the survey revealed the most and least attractive factors when choosing an internship place. The highest priority factors are the salary level (348 respondents) and training in a company (442 respondents) (Fig. 5 and Table 2). The lowest factors include the level of the company’s scale and the possibility of relocation to another city or country (Fig. 5 and Table 2). For the integrated evaluation of value propositions, we used the Pareto diagram, which is common in the field of personnel management and allows identifying 20 % of the most significant factors that give the employer brand an 80 % success rate (Fig. 6). Unlike [15], where company Deloitte, USA identified only general changes in the values and behavior of Generation Z, analysis conducted in this study enables an employer to form a better value proposition for Generation Z. Thanks to this analysis, the description of internship positions will be more attractive for candidates. Due to the global pandemic, there have been changes in HR processes in companies. First of all, it is the work format of the company’s employees. Most of them were transferred to a remote work schedule in order to avoid the spread of morbidity. The hybrid schedule turned out to be most convenient for Generation Z, even in the absence of quarantine restrictions (Fig. 8). This is a work format of employees, in which they work three days in the office and two – remotely (Fig. 8). Unlike [15], where there is no information about the work format as a component of the value proposition of the employer brand, this allows creating new value propositions during the formation of the employer brand for Generation Z, which can be effectively used by companies not only in the context of a global pandemic but also when designing changes in HR processes in companies. The complex comprehensively considers and identifies the main value propositions that are of the most and least priority for Generation Z. When choosing value propositions, modern labor market trends, digitalization of the economy, the COVID-19 pandemic were taken into account. The drawback of the study is the lack of open questions. Elimination of this drawback would lead to an increase in the difficulty of processing survey results, which is not justified. Further research will be aimed at the construction of a methodological toolkit for evaluating the employer brand and organization of management activities for its formation by enterprises. 7. Conclusions 1. According to the results of the survey, the main modern trends of the employer brand in the labor market were analyzed, indicating their rating of attractiveness for Generation Z. Among the 9 leading retail companies, “Rozetka”, “Fozzy Group” and “Comfy” were the most attractive for Generation Z, according to the results of the survey. Among 13 leading companies in the FMCG sphere, “Nestle” and “Coca-Cola HBC Ukraine” were the most attractive for Generation Z. This allowed revealing the best companies that use the employer brand when searching for staff, and whose experience can be used as best management practices. 2. Based on the results of the study, it was found that in order to form the employer brand, it is necessary to take into account and balance the qualities and characteristics of potential candidates for employment. In accordance with this, the employer brand should be considered from the point of view of the requirements of an enterprise for them and the value proposition of companies in the labor market. Generation Z as potential employees is attractive to today’s employers, who expect their employees to have creativity, energy, and opportunities to learn and develop with the company. However, to do this, companies need to form specific value propositions that would satisfy this target audience. With the help of established value propositions, companies position themselves in the labor market and create their own image for potential employees. A list of the main value propositions that are important for Generation Z when choosing a place of work and internship was formed. As a result of the survey, the priority of value propositions for Generation Z choosing a place of work and internship was determined. The 89 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 following value propositions received the greatest importance for choosing a place of work: salary level and training in the company. The possibility of relocating to another city/country and the company’s business rating were of the least importance. The following value propositions turned out to be most important for choosing an internship: salary level and training in a company. The possibility of relocating to another city/country and the company’s business rating were the least important. Internships are an important part of staff selection; it makes it possible to accurately identify the most suitable employees for a company. On the one hand, potential employees should understand and evaluate the would-be employment. On the other hand, employers do not want to spend extra money and time searching for the necessary personnel. The study of the priority of the factors, considering which representatives of Generation Z decide to stay in the company after an internship showed tangible and intangible incentives that guide them. The most important are: the salary level and training in the company. The least important are the possibility of relocating to another city country and the company’s business rating. This indicates a certain balance of tangible (salary) and intangible (training opportunities) value propositions when choosing a company both for internships and for work after an internship. Openness, 1/13 ( 115 ) 2022 flexibility, and dynamism of the behavioral characteristics of Generation Z representatives make the value proposition to relocate to another city/country unimportant for them. And the absence of authority leads to the low priority of business rating and the reputation of a company. 3. Quarantine restrictions significantly affected the organization of work with the staff and the search for job candidates. The study showed which factors are most attractive to Generation Z under conditions of remote work. Thus, the most attractive is the schedule in which employees work three days in the office and 2 days remotely. The least attractive is the schedule when employees work remotely 5 days a week. This indicates the desire of representatives of Generation Z to plan their time independently, to have spare time for development and training in attractive areas of interest. The content and the level of significance of value propositions for Generation Z will allow companies to better understand their target audience when forming their own employer brand and focus on the necessary aspects of managing their present and would-be staff. 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(2015). The Study and Formation of the Employer Brand: Theoretical and Applied Aspects. Problemy ekonomiky, 4, 247–252. Available at: http://nbuv.gov.ua/UJRN/Pekon_2015_4_34 23. Urbancová, H., Hudáková, M. (2017). Benefits of Employer Brand and the Supporting Trends. Economics & Sociology, 10 (4), 41–50. doi: https://doi.org/10.14254/2071-789x.2017/10-4/4 24. Wojtaszczyk, K. (2012). Rozdział 2. Employer branding w kontekście ewolucji nauki o zarządzaniu i przeobrażeń wewnątrz organizacji. Employer branding czyli zarządzanie marką pracodawcy. Łódź: Wydawnictwo Uniwersytetu Łódzkiego, 47. 91 

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Qeios · Definition, February 2, 2020 Ope n Pe e r Re v ie w on Qe ios Progesterone Receptor Positive by Immunohistochemistry 31-40 Percent National Cancer Institute Source National Cancer Institute. Progesterone Receptor Positive by Immunohistochemistry 3140 Percent. NCI T hesaurus. Code C141451. An immunohistochemical staining finding indicating that 31-40 percent of the cells in a tissue sample are expressing progesterone receptor. Qeios ID: E09RIS · https://doi.org/10.32388/E09RIS 1/1 

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3 Customer Challenges in Times of Global Risk and Uncertainty Tony Carter, Demissew Ejara, Christina Reis and Walter Carter University of New Haven, New Haven U.S.A. 1. Introduction Rapidly changing business conditions have made it more difficult and challenging for managers to keep customers (Carter, 2008). To build this process it is necessary to consult customers for preferences, build familiarity and knowledge to build a relationship and conduct business in a customized fashion. The process takes every opportunity to build customer satisfaction with each customer contact. It is an important process to have, since customers today are more demanding, sophisticated, educated and comfortable speaking to the company as an equal (Belk, 2003). Customers have more customized expectations so they want to be reached as individuals (Raymond and Tanner, 1994). Also, a disproportionate search for new business is costly. The cost to cultivate new customers is more than maintaining existing customers (Cathcart, 1990). Other reasons that Customer Retention is necessary is because many unhappy customers will never buy again from a company that dissatisfied them and they will communicate their displeasure to other people. These dissatisfied customers may not even convey their displeasure but without saying anything just stop doing business with that company, which may keep them unaware for some time that there is any problem (Cathcart, 1990). 2. Causes of crisis Poor customer relationship building can be a cause for crisis. About 91% of unhappy customers will never buy again from a company that dissatisfied them and they will communicate their displeasure to other people. There can be various reasons that customers become dissatisfied with a company. It can be on the basis of product or service quality, price, poor location, lack of attentiveness, complacency, not having a customer complaint system or a weak public image. In addition, about one third of all customers are dissatisfied because they were unappreciated and some of those customers will never complain to the company. Companies that offer employees a sense of long-term stability may satisfy customers and prosper at the expense of less effective competitors. So, the failure of managers to have a process to monitor and meet customer satisfaction needs can be devastating to a company. One particular cause of crisis is that senior executives too often do not understand the fundamentals of their business. They neglect to ask central questions, such as what precisely is their company’s core expertise, what are reasonable long and short-term goals, what are the key drivers of profitability in their competitive situation. www.intechopen.com 40 The Economic Geography of Globalization The occurrence of a crisis and its aftermath can have a devastating effect on a company’s sales. For every $100 million in revenue, two incidents per year occur that call on a company’s emergency response plan. For every $8 billion of revenue, there is one major loss per year, representing about one percent of annual sales. One catastrophic loss every 10 years, equals 1.5 times annual profit. Why do corporations fall short of objectives? Why do strategies that seemed eminently sensible turn out to be disasters? Just why do successful organizations, which once could do no wrong, suddenly begin to lose their way? As seen from the Barings Bank financial debacle, the role of sales management is crucial in today’s global business arena role. Nick Leeson, as the trader who caused the Baring’s Bank scandal in London, essentially performed in a sales role. To guard against billion dollar catastrophes and a host of lesser risks, the best line of defense is a solid crisis management process. 2.1 The consequences of crisis Crisis can be characterized as a major unpredictable event that has potentially negative results. It can also be thought of as a turning point for better or worse. The crisis event and its aftermath may significantly change a sales organization and its salespeople, products, services, financial condition and reputation. So, the real consequences of crisis events to managers can mean the loss of future sales and reputation, the loss of consumer confidence, prolonged negative publicity, exposure to lawsuits, declines in stock values and increases in operating expenses. 2.2 Union Carbide In the area of crisis management, few firms have experienced more than Union Carbide. In 1984, gases released from a pesticide plant of an affiliate — owing to a deliberate act of sabotage — killed about 2,000 people in Bhopal, India, and injured many more. The “Crisis” as such obliged the company and its Indian affiliate to pay $470 million to the Indian government to compensate victims and survivors. The “Crisis” also ultimately depressed the stock price so much that they were subject to an unfriendly takeover attempt. To fight it off, they had to sell off significant portions of the company and use the proceeds to pay a special dividend to the stockholders. To forestall another tragedy of such proportions, Union Carbide’s management resolved to create the world’s best episodic risk-management system (ERMS), one that ensures senior executive review of substantial risks. This incident really indicates what can be at stake with a crisis. Starting in 1985, Union Carbide spent five years building a database of information that catalogs hazardous materials stored on site, size of storage tanks, vulnerability of local people to an explosion, and so on. Arthur D. Little, Inc. of Cambridge, Massachusetts, weighed the variables and ranked every one of the company’s 1,400 operations. So, when the company initiated its ERMS program, responsibility was assigned to the senior line managers, not the risk manager. The company spent between $5 million to $10 million to develop its ERMS and spends about $1 million a year to manage it. Another customer-tracking technique that can help prevent a crisis is considering the needs of customers all along the value chain, not just the end user. Every company must please the whole series of customers and target audience. Depending on the business this can be consumers, wholesalers, shippers, retailers, independent distributors, employees, stockholders and the financial community. By failing to meet the “customized” needs and www.intechopen.com Customer Challenges in Times of Global Risk and Uncertainty 41 expectations of their customers, companies will find that over time they will lose these customers. 2.3 The success of Rohm Co. Rohm Co., a Japanese firm, earned a record $267 million in the fiscal year ended March 31, on revenues of $2.8 billion. Its 17% pretax earnings margin compares with around 4% for Japan’s electronics industry as a whole. Rohm is Japan’s leading producer of laser diodes, semiconductors that function like phonograph needles in compact disc players. Rohm is also the leading producer of the chips used in computer floppy disk drive motors. In a country where businessmen often wait for crisis to strike and then react, Rohm’s management has demonstrated an ability to anticipate crisis. Most important, their managers assess the sales effort rigorously, even ruthlessly. Rohm’s after-tax profit margins were slipping into the 2% range, which is not drastically low by Japanese standards. That same year Sony’s net margins were around 3%, but Rohm’s management was thinking ahead concerning what would happen to margins if sales started to slip? Not content to wait for answers, Rohm’s management shook up the company. They forced Rohm’s salesmen to refuse unprofitable and low-margin orders. This was almost unheard of in Japan, where suppliers tend to do just about anything to please a customer. As a result of the changes, Rohm’s sales rose by 21%, after tax margins were 9% and profits will grow about 10% annually over the next 3 years. 2.4 Summary Success with customers in business globally, must be viewed as an evolutionary process. Growth within an organization stems from a few basic core strategies that result in maximized profit. For example, In Vilnius, Lithuania, outdoor common food vendors advertise their unique products by offering free samples of any food item customers are interested in. In Moshi, Tanzania, E. Africa, the Chaga tribe markets their freshly brewed banana beer by placing a branch of the yucca plant on the ground outside and in front of their brewery. This lets fellow Chaga tribe members know that freshly brewed banana beer is inside. The validity and appropriateness of risk policies need to be re-evaluated constantly. In addition, organizations must develop customer relationship building, have interaction with top executives from top customers, help customer satisfaction and loyalty, encourage top management to develop, a crisis management culture and philosophy that permeates the company. 3. Other causes of crisis: the global crisis 0f 2007-2009 An economy is said to be in crisis often during the recession or depression stages of the business cycle. The basic characteristics of economic crisis are slow down in production and distribution of goods and services, increase in unemployment due to layoffs, decrease in consumer confidence and hence expenditure, decrease in availability of credits, etc. An economic crisis can be caused by several factors. The 2008 global economic crisis is referred to as financial crisis because it started in the financial services sector. The trigger of the crisis is decline in the market prices of houses. The Standard and Poors’ Case-Shiller home price index showed an aggregate decline in home prices of more than 20% since 2007. Why would decline in home prices cause global financial crisis instead of just crisis in the real estate sector? Three factors contributed to the escalation of the housing crisis into global www.intechopen.com 42 The Economic Geography of Globalization financial crisis. These are (1) the loosened financial regulation, (2) the increasing securitization of loans including mortgage loans, and (3) the expectation that house prices would continue to increase. 3.1 The regulatory environment In 1933, the US Congress passed the Glass-Steagall Act that separated commercial banking, investment banking and insurance services. The objective of the Glass-Steagall Act was to minimize conflict of interest when the same institution acts as lender as well as securities broker. In 1999, Congress passed the Gramm-Leach- Bliley Act that repealed the GlassSteagall Act. This allowed commercial banks to engage in investment banking activities. For example Citicorp, a commercial bank expanded into investment banking by acquiring Smith Barney, an investment bank. Subsequently, it merged with Traveler’s Insurance, which already acquired Solomon Brothers, and formed CitiGroup, a bank holding company with multiple financial services1. The trend seemed toward increasing integration of financial services industry and the creation of multi-tasked one-stop-shop companies. There are two main reasons for the repeal of the Glass-Steagall Act. First there is no such law in other countries. Increasing globalization of the financial services industry put the US companies at a competitive disadvantage. Second, there was pressure to deregulate the financial services on the assumption that the companies themselves put in place adequate risk management systems. This assumption is based on the increasing application of computer systems and complex mathematical models in the design and implementation of risk management systems. These facts not only loosened regulatory rules but also reduced the ability of bank regulators to enforce the existing rules. Financial institutions were able to classify complex assets to suit their purpose. One of the safety and soundness regulatory tools is risk based capital adequacy requirement. Bank assets are classified into risk categories and risk weights assigned with risky assts having higher weights. The risk-weighted total asset is the basis for determining capital adequacy. The Basel Committee on Bank Supervision proposed a minimum capital requirement of 8%2. This ratio has been implemented in the US for many years. Maintaining high level of equity capital is good for safety of the financial institution. But its return on equity will be enhanced if the bank is financed with a greater proportion of debt. The return on equity is related to the proportion of capital by the following formula: ROE = ROA*A/E Where ROE = Return on equity (to the shareholders) ROA = Return on assets (return on total investment) A/E = ratio of assets to equity, which is the reciprocal of capital ratio. For example, if a bank earns a rate of return on assets of 4% and its capital ratio is 8%, its return on equity will be 4%*1/0.08 = 50%. If the capital ratio is decreased to 5%, the ROE would increase to 4%*1/0.05 = 80%. So the motive of the banks not to invest more of own The mergers actually took place in 1998, before the repeal of the Glass-Steagall Act in 1999. The merger was allowed on the condition that Citi would divest some of the divisions within the following five years. But the passage of the Gramm-Leach-Bliley Act rendered enforcement of the divestment unnecessary. 2 Basel Committee on Banking Supervision (2004) “International Convergence of Capital Measurement and Capital Standards.” (http://www.bis.org/publ/bcbs107.htm). 1 www.intechopen.com Customer Challenges in Times of Global Risk and Uncertainty 43 equity capital is to lever up profitability by using debt to finance operations. But low capital ratio reduces the capital cushion and increases risk. Just like the bank assets are grouped into risk categories, bank capital is also grouped into two tiers. Tier 1 capital is made up of contributed common stock plus retained earnings. Tier 2 capital is composed of preferred stocks and some subordinated long-term debentures. With the complexities of the securities the banks issue directly and through their brokerage arms and the assets in which they invest, it is difficult to enforce capital adequacy rule satisfactorily. Banks created special purpose entities for the purpose of carrying risky assets on their balance sheets instead of on the balance sheets of the banks. Profits from such risky assets accrue to the banks through intercompany transfers of profits but since the banks are not maintaining adequate capital and regulators find it hard to implement capital adequacy requirements based on off-balance sheet assets, the risk would be very high. It is a high risk game with inadequate cushion for emergency. When house prices declined in 2008 and the mortgage loans became non-performing, many banks went bankrupt or were taken over at fire-sale prices. 3.2 Securitization Securitization is a process by which a tradable security is created and issued supported by a pool of other assets. The tradable security so created is sometimes referred to as asset backed security (ABS). The pool of assets that support the ABS could be any financial instruments with streams of cash flows. A typical example is the securitization of mortgages. ABSs supported by a pool of mortgage loans are known as Mortgage Backed Securities (MBS). Securitization of mortgages helps increase the volume of funds available to finance home purchases. Financial institutions can issue the MBS in the capital markets, raise more funds and lend to finance more real estate purchases. Fannie Mae and Freddy Mac are instrumental in the purchase of mortgages and their securitization. Securitization also bundles assets of different risk categories. For example some sub-prime mortgages may be bundled together with high quality mortgages to create an MBS. Such MBSs are very risky even if the proportion of sub-prime loans included is small. The objective of the banks in securitization is to remove the loans from their balance sheets. Loans are assets for banks. Capital adequacy regulation requires banks to maintain capital based on risk-weighted assets. Loans have higher risk weights than cash for capital adequacy purposes. So securitization and removal of loans from bank balance sheets help them reduce the amount of capital they need to maintain. In many cases, the banks transfer the loan to their securities brokerage arm and in some cases, they establish special purpose entities that finance the securities purchase with commercial paper issues of their own3. The funds raised by selling MBS are then used to finance more home purchases, which are in turn securitized, and the expansion continued. According to Acharya and Richardson (2009), securitization worldwide went from $767 billion at the end of 2001 to $1.4 trillion in 2004 to $2.7 trillion in 2006. Although there are AAA rated tranches in the MBS, there are many which are supported by sub-prime mortgages. The rating agencies are accused of giving high ratings to many sub-prime supported MBS because of self interest. These mortgages also have different sub-categories. Some are fixed rate conforming mortgages where periodic payments by the borrower 3 Acharya, V.V. and Richardson, M. (2009) “Causes of the Financial Crisis.” Critical Review, 21(2-3) 195210. www.intechopen.com 44 The Economic Geography of Globalization include interest plus principal. There are also variable rate mortgages, where interest rate is variable for the first several years and then reset to a fixed rate after that. There are also interest only mortgages, where the borrower pays interest only for the first several years and then interest plus principal repayment starts thereafter. The increase in supply of loans for home purchases due to securitization increased home purchases. Real estate prices also rose resulting in what is referred to as the house price bubble. Many people cashed the equity on their homes. Banks were attracted by the profitability of mortgage loans that could be securitized and removed from the balance sheet and increased lending liberally. The relatively low interest rates of the 2001-2003 and the expectation that home prices would continue to increase attracted both the lenders and the borrowers. In 2006, interest rates started rising and many of the adjustable rate and interest only mortgages were ready to reset to a fixed rate. At the new high interest rate, many home owners couldn’t afford to pay their new monthly obligations. Default rates increased and the demand for houses decreased. Banks could not sell repossessed homes at high enough prices to recover their loans. With huge amount of outstanding mortgages that were securitized and put on the balance sheets of special purpose entities, with banks that were not adequately capitalized, and with decrease in real estate prices, the stage was set for the bubble burst and the financial crisis. Many banks tightened credit, which made refinancing difficult and further reduced home prices. This in turn decreased consumer confidence and consumer expenditure. Consumer expenditure accounts for about one third of the US economy. Decrease in expenditures decreased demand for goods and services and the crisis spread to all the sectors of the economy. Businesses reduced inventory restocking in the event of decreased demand, and also reduced expenditures on machinery and equipment investments. Decrease is sales affected companies across the economy. Banks wrote off billions of dollars worth of MBS and other real estate related loans. Many companies filed for bankruptcy. Business bankruptcy filings increased from 4,086 in the first quarter of 2006 to 16,014 in the second quarter of 20094. 3.3 House price expectations The third factor that contributed to the crisis is the expectation of continuous increase in home prices. With the increase in the supply of real estate loans and liberal credit policies, home prices increased continuously. According to FHA home price index data, home prices more than doubled from the early 1990s to 2006. See figure 1. As Figure 1 shows, house price increase accelerated after 2001 and surpassed the GDP growth. Such increase in real estate prices enticed banks to lend liberally with the assumption that any mortgage defaults would be recovered by repossessing the property and selling it at higher price than the loan balance. Many of these loans are sub-prime loans with various liberalization incentives such as variable rate mortgages and interest only mortgages. These mortgages usually reset to fixed rate in five years time. The growth in home prices started stabilizing in 2006 and reached a peak in the second quarter of 2007. Then the decline started triggered by reset of mortgage rates to a fixed and a higher level, which caused defaults. Banks tightened credits. Businesses and consumers lost confidence and decreased expenditures. These decreased the overall economic activities. 4 American Bankruptcy Institute (http://www.abiworld.org). www.intechopen.com Customer Challenges in Times of Global Risk and Uncertainty 45 Fig. 1. Comparison of Real GDP, home prices and stock market index in the USA from first quarter of 1991 to the third quarter of 2009. The graphs represent the three indexes rescaled by setting 1991.1 to 100 to facilitate comparison. GDP data are obtained from Bureau of Economic Analysis (http://www.bea.gov), home price index data are obtained from Federal Housing Finance Agency ( http://www.fhfa.gov/Default.aspx?Page=87) and the stock price index data are from Morningstar EnCorr database As Figure 1 shows, decline in house prices preceded decline in real GDP and decline in the stock prices. Clearly, the cause of the crisis is the decline in house prices. Stock indices lost about half of their pre-crisis value. 3.4 Consequences Financial institutions specifically got stuck with huge amounts of illiquid MBS that they couldn’t sell. Many wrote them off. Some couldn’t sustain the losses and made strategic moves such as merger with other companies. Bear Sterns, Merrill Lynch, Countrywide Financial and Wachovia were taken over by other financial institutions. Lehman Brothers went bankrupt. JP Morgan Chase and Morgan Stanley became bank holding companies in the hope that they could attract deposits and get access to the Federal Reserve’s Discount Window facilities. Fannie Mae and Freddie Mac, the two government-sponsored entities established to provide home financing through the purchase of mortgages, were taken over by the government. The US Government spent billions of dollars to bail out banks and insurance companies. American International Group (AIG) and Citigroup were saved by government bailout. Many other regional financial institutions had to make similar strategic moves. Some went bankrupt and others were taken over by other stronger institutions. Outside the financial services industry, the auto industry was severely affected. The three major auto makers sought government assistance to avoid bankruptcy. General Motors and www.intechopen.com 46 The Economic Geography of Globalization Chrysler obtained massive government assistance in the form of loans or acquisition of preferred stocks. Still General Motors filed for Chapter 11 bankruptcy protection. Several other companies in the auto industry were affected. Lear Corporation, supplier of car seats, filed for Chapter 11 bankruptcy protection and wiped out original shareholders’ value and reorganized by issuing new shares. The main cause of weakness in the auto industry is also decrease in consumer expenditure. In addition, the US automakers are not up to date in terms of innovation compared to European and Japanese auto makers. The retail industry also suffered a great deal. Retail sales declined by up to 12% because of decrease in consumer confidence and hence consumer expenditures. Consumer confidence index fell below 30 in the first quarter of 2009 (see Figure 2). Consumer confidence below 50 indicates recession (contraction). When the banks restricted credit, some of the weaker retail businesses couldn’t sustain and went bankrupt. Examples include Circuit City Stores, Linen and Things, and Bernie’s. These companies couldn’t secure financing from banks and neither could they attract any acquirer. Blockbuster tried to buy Circuit City, but it withdrew the offer after getting access to the latter’s books during the due diligence investigation. Many people also thought it would be a strategic mismatch to merge Circuit City with Blockbuster. Fig. 2. Percentage change in retail sales (left scale) and level of consumer confidence (right scale) 2007 – 2009. Source: http://www.bloomberg.com/markets/ecalendar/index.html The hi-tech industry suffered loss of sales and loss of value, but there are no major bankruptcies. The industry suffered decline in sales due to decline in expenditures by individuals as well as businesses. Most of the computer makers and software developers had adequate cash on hand that helped them withstand the adverse consequences of the recession. But there have been some consolidations with relatively stronger companies acquiring the relatively weaker ones. For example, Oracle took over BEA Systems in 2008 and Sun Microsystems in 2009. Microsoft Corporations bid for Yahoo, Inc. faced resistance www.intechopen.com Customer Challenges in Times of Global Risk and Uncertainty 47 and failed, however. The decline in stock prices increased merger and acquisition activities in other industries too. In general, the crisis started in the financial services sector spread to all the other industries and across the globe and resulted in loss of confidence, loss of sales to businesses, loss of wealth to investors, loss of jobs and bankruptcy of many companies. Unemployment rate exceeded 10% in the third quarter of 2009, a record for several decades. Stock markets lost about half of their value. 3.5 Government reaction The US Government reacted to the crisis swiftly and on a scale unprecedented in recent memory. The Department of the Treasury, the Federal Reserve Bank and the Federal Deposit Insurance Corporation (FDIC) are the main government agencies directly involved. All the three made significant changes in terms of both the magnitude and nature of their involvement in the economy. Department of the Treasury: The Department of the Treasury developed the Troubled Assets Relief Program (TARP) through which it engaged in outright bailout of companies in the form of lending, acquisition of securities and guaranteeing of loans. The initial capital set for the TARP amounted to $800 billion. As of September 2009, $364 billion of this capital had been disbursed. The major recipients are AIG5 ($70 billion), Citigroup ($45 billion), Bank of America6 ($45 billion), General Motors Corporation7 ($49.5 billion), Wells Fargo ($25 billion), JP Morgan Chase ($25 billion), Goldman Sachs ($10 billion), Morgan Stanley ($10 billion) and several regional banks8. These assistances came with restrictions on the part of the companies in the form of restrictions on executive compensation, dividend payments, corporate expenses and other measures. Banks could repay these loans only after passing a stress test assessment, which included ability to raise debt and equity capital in the financial markets without government guarantees. The lions’ share of the Treasury’s assistance went to the financial services industry because they wanted the banks to release funds to businesses and individuals in the form of increased credit facilities and reinvigorate the economy. But assistance also went to the auto industry and local governments. In addition to loans and investment in companies, the Treasury undertook consumer and business lending initiatives. Tax credit of up to $8,000 is offered to households who purchased homes in 2008 and 2009. The cash-for-clunkers program offered credit for 5 Treasury’s assistance to AIG took the form of $40 billion purchase of preferred stock and about $30 billion loan facilities, out of which AIG drew only about $3.2 billion. AIG received additional credit facilities of up to $85 billion from the Federal Reserve Bank. 6 This sum represents preferred stock purchases of Bank of America and Merrill Lynch in October 2008 of $15 billion and $10 billion respectively. After Bank of America completed it acquisition of Merrill Lynch, the Treasury purchased an additional $20 billion preferred stock in January 2009. 7 These include a total of $19.4 billion loans before GM filed for bankruptcy on June 1, 2009 and $30.1 billion debtor-in-possession loans with the bankruptcy. GM filed for bankruptcy restructuring on June 1, 2009 and subsequently reorganized under a New GM. Old GM common equity was wiped out. Treasury’s loans to the Old GM were transferred to new GM in the form of preferred stock and common stock. The US Government owns about 61% of the New GM. 8 These figures represent aggregate amounts disbursed under different programs. Some are loans and some are Targeted Investment Programs. See the details in United States Department of the Treasury, Office of Financial Stability “Agency Financial Report, Fiscal Year 2009” available at http://www.treas.gov/press/releases/OSF%20AFR%2009.pdf. www.intechopen.com 48 The Economic Geography of Globalization trading-in less gas efficient cars for more gas-efficient cars for a limited period during 2009. Both programs were popular and received high publicity. The results have been encouraging. The economy started recovery in mid 2009. Real GDP grew by an annualized 4.6% during the third quarter and 5.7% during the fourth quarter of 2009. By the end of 2009, several of the financial institutions that received government assistance repaid the loans and redeemed the preferred stocks. These include Goldman Sachs, JP Morgan, Morgan Stanley, and Bank of America. The Federal Reserve Bank: As the monetary policy maker of the country, the Federal Reserve Bank controls money supply, regulates financial institutions and acts as bank of banks. Its monetary policy tools are the federal funds rate, an overnight interest rate at which banks borrow from each other, open market operations through which it monitors amount of money in circulation, reserve requirement on commercial banks deposits, and discount window facilities, through which it lends short-term funds to commercial banks. The Fed increases the federal funds rate when there is fear of increased inflation and decreased the federal funds rate when there is fear of economic contraction. The Fed enforces its interest rate policy through open market operations. If it buys Treasury securities thereby releasing more currency into circulation, it reduces interest rate, and if it sells Treasury securities thereby reducing the amount of money in circulation, it increases interest rate. Buying Treasuries is expansionary monetary policy and selling Treasuries is contractionary monetary policy. Thus the size of the balance sheet of the Federal Reserve shows whether it is following expansionary or contractionary monetary policy. The assets of the Federal Reserve include securities it purchased, loans to financial institutions and governments, and any gold and foreign currency reserves. Its liabilities are currencies in circulation and deposits of financial institutions and government agencies. To mitigate the impact of the financial crisis, the Fed obviously followed an expansionary monetary policy. The magnitude and the composition of its balance sheet changed tremendously. As Figure 3 shows, total assets of the Federal Reserve increased from around $870 billion in August 2007 to about $2.24 trillion in December 2009. The biggest jump was made in September 2008. This is the largest expansion in the history of the Fed and resulted from the Fed’s attempt to fight the recession. The composition of its assets also changed.9 Traditionally, the Fed bought and sold only short-term Treasury securities and overnight secured agency debts in the form of repos. This time, its purchases included long-term Treasuries and government agency securities with up to ninety day maturity. The Securities held outright in Figure 3 represent Treasuries as well as agency and agency-guaranteed MBS and loans extended to AIG. These agencies are mainly Fannie Mae, Freddie Mac and Ginnie Mae. These securities holdings increased from around $500 billion during the pre-crisis period to over $1.90 trillion in 2009. The Fed also extended short-term liquidity facilities to support commercial paper loans, collateralized debt obligation (CDO) loans, and other term loans. These facilities are new and amounted to over $1.6 trillion in the late 2008 and early 2009. These loans focused on large companies with systemic impact on the economy. Traditionally, the Fed acted as lender of last resort for depository institutions. Lending to some non-depository institutions was a departure from this traditional policy. These loans gradually decreased as the economy recovered and repayments are made on the loans. 9 For a brief discussion of the Fed’s actions and the composition of its lending facilities, see Statement by Ben S. Bernanke for the Committee on Financial Services, US House of Representatives, February 10, 2010 available at http://www.federalreserve.gov. www.intechopen.com Customer Challenges in Times of Global Risk and Uncertainty 49 All Liquidity Facilities includes: Term Auction credit; primary credit; secondary credit; seasonal credit; Primary Dealer Credit Facility; Asset-Backed Commercial Paper Money Market Mutual Fund Liquidity Facility; Term Asset-Backed Securities Loan Facility; Commercial Paper Funding Facility; support for American International Group, Maiden Lane LLC, Maiden Lane II LLC, and Maiden Lane III LLC, and central bank liquidity swaps. Securities held outright include Treasury, agency, and agency-guaranteed mortgage-backed securities under the large scale asset purchase program announced by the Fed. Fig. 3. Total Assets and selected assets of the Federal Reserve Bank (2007-2009). Figures are in millions of dollars. Source: The Federal Reserve website (http://www.federalreserve.gov/monetarypolicy/bst_recenttrends.htm) In addition to the expansion of its balance sheet, the Fed decreased the federal funds rate to an unprecedented level. In 2008, the federal funds rate was reduced to 0-0.25% range and it has been kept that way since then. The federal funds rate is a conduit that influences the general level of interest rate in the economy. The objective of the Fed in keeping the federal funds rate so low is to encourage banks to lend and businesses and individuals to borrow and invest and speed up the economic recovery. Federal Deposit Insurance Corporation: If a bank fails, the available cash is distributed to the depositors on a first-come first-serve basis. So depositors have to act fast to get their money. Then the depositors will lose confidence in the banking system and start withdrawing their deposits even from healthy banks and cause bank run. To minimize such bank runs, the Federal Deposit Insurance Corporation (FDIC) guarantees deposits up to $100,000 per depositor in each insured bank. If a bank is in trouble, the FDIC either bails it out or let it fail and pays depositors up to a maximum of $100,000. With the advent of the financial crisis, many banks failed. The number of bank failures was exceptionally high compared to other years (see the table of bank failures). In 2008, 25 banks failed and in 2009, 140 banks failed. FDIC facilitated the takeover of some of these banks by other relatively healthy ones. Merger with other healthy banks were facilitated by FDIC in cooperation with the Federal Reserve and the Department of the Treasury. To mitigate the probability of depositors causing bank runs, the FDIC increased the deposit insurance limit to $250,000 per depositor in 2008. This limit increase will last until the end of www.intechopen.com 50 The Economic Geography of Globalization 2013. In addition, the FDIC implemented a temporary liquidity guarantee program in 2008 to provide liquidity to banks and also guarantee some loans. Year Count 2001 4 2002 11 2003 3 2004 4 2005 0 2006 0 2007 3 2008 25 2009 140 2010 15* Total 205 *2010 frequency is for the month of January only. Table 1. Frequency of bank failures by year (2001 – 2009) closed by the FDIC Source: Compiled based on data from the web site of FDIC at http://www.fdic.gov Such coordinated effort and the scale of intervention by the government is unprecedented. The efforts appear to be successful for the stock market gained about 40% during the last three quarters of 2009 and real GDP increased during the third and fourth quarters. The efforts saved the economy from falling into the scale of the Great Depression. With the increased liquidity in the economy, there is fear of increased inflation in the near future. The Fed may have to reverse its policy and tighten monetary policy to fight such fears. 3.6 Crisis management at firm level What should companies do to minimize the impact of crisis on their performance? Crisis of the 2007-09 proportion, affect every company. Some companies do not survive while others emerge stronger. What are the basic characteristics of companies that survive the crisis and what are the basic characteristics of those that don’t survive the crisis? Two basic features stand out when we compare the survivors and the vanquished. These are having good fundamentals and maintaining adequate liquidity. Good fundamentals: companies with good fundamental financial positions have the strength to withstand adversities in the event of crisis. Companies with poor fundamentals will find it harder to get credit and maintain adequate customer base. In the 2007-09 crisis, financial institutions tightened credit across the board. Companies with poor financial positions find it harder to get the usual credit because they are not qualified based on the new stringent criteria the banks have established. For example, Bernie’s is liquidated for this reason. Companies with good fundamental financial positions may not be desperate for external financing and even if they seek external financing, they qualify. The following tables present comparisons based on fundamentals for selected group of companies. The companies selected for comparison are in the same industry. For example Best Buy and Circuit City Stores are competitors and the former survived and the latter went bankrupt. www.intechopen.com 51 Customer Challenges in Times of Global Risk and Uncertainty Panel A. Revenue Growth of selected companies: 3-Year compound annual growth rate (%) Company Name 2000 2001 2002 2003 2004 2005 2006 2007 2008 BANK OF AMERICA CORP 38.615 0.866 -3.459 -5.399 7.183 22.422 33.152 22.119 9.963 COUNTRYWIDE FINANCIAL CORP* 25.504 16.205 50.54 40.863 30.399 19.099 20.948 21.986 n.a. WACHOVIA CORP* 16.883 1.303 2.225 2.269 7.814 15.031 24.13 25.537 n.a. WELLS FARGO & CO 41.979 11.478 10.273 4.875 6.085 11.403 14.694 16.521 8.528 BEAR STEARNS COMPANIES INC* GOLDMAN SACHS GROUP INC LEHMAN BROTHERS HOLDINGS INC* MERRILL LYNCH & CO INC* MORGAN STANLEY BED BATH & BEYOND INC LINENS N THINGS INC* BEST BUY CO INC CIRCUIT CITY STORES INC* 19.137 2.926 -4.381 -10.387 -1.167 18.796 30.805 24.349 n.a. 17.326 11.475 -3.413 -10.545 -1.41 23.826 43.189 43.388 7.283 16.138 4.022 -4.037 -13.214 -1.73 24.547 39.282 40.552 n.a. 12.244 2.622 -6.893 -14.807 -5.604 19.286 35.236 24.514 -29.482 18.732 11.992 -1.685 -8.374 -3.292 17.333 29.889 29.217 3.524 30.978 27.968 24.969 23.166 20.693 16.596 13.903 11.046 21.618 19.595 18.872 15.057 13.426 n.a. n.a. n.a. 22.399 24.872 18.795 9.357 Panel B. Return on Investment (%) Company Name 2000 BANK OF AMERICA 7.446 CORP COUNTRYWIDE FINANCIAL CORP* 3.197 WACHOVIA CORP* 0.285 WELLS FARGO & CO 7.933 BEAR STEARNS COMPANIES INC* GOLDMAN SACHS GROUP INC LEHMAN BROTHERS HOLDINGS* MERRILL LYNCH & CO INC* www.intechopen.com 17 11.865 13.774 13.545 13.417 13.425 0.89 -1.87 -2.216 2.996 5.009 8.327 3.875 2001 2002 7.456 n.a. 2003 2004 2005 2006 n.a. 2007 2008 4.715 0.635 2.299 3.373 4.943 5.116 4.737 5.271 -1.261 2.472 5.355 6.363 5.674 6.634 4.431 3.167 6.128 8.996 7.224 7.241 7.528 7.171 6.264 n.a. n.a. 0.758 6.467 8.459 9.724 7.506 8.602 7.994 3.206 2.248 3.274 3.451 3.319 3.009 3.375 0.3 n.a. 6.905 4.691 3.663 3.738 4.232 4.276 4.959 4.608 0.811 4.551 2.982 2.245 3.211 3.508 3.975 3.826 2.805 n.a. 5.361 0.696 3.02 4.105 3.361 3.402 3.929 -3.822 -16.922 52 The Economic Geography of Globalization MORGAN STANLEY 10.919 5.805 4.532 4.575 BED BATH & BEYOND INC LINENS N THINGS INC* 21.043 20.067 20.812 20.066 22.914 25.32 22.431 21.969 14.169 14.148 5.971 10.355 9.823 7.478 n.a. n.a. n.a. n.a. BEST BUY CO INC CIRCUIT CITY STORES INC* 4.07 4.124 4.612 1.306 0.881 19.763 17.097 17.482 20.492 18.766 20.975 20.173 27.315 15.966 6.517 7.411 1.767 -0.035 2.854 7.531 -0.553 -20.597 n.a. *company did not survive the crisis. It either went bankrupt or is taken over by other company at low price. Table 2. Comparison of Fundamentals for Selected Companies Source: Compiled based on data from Research Insight Table 2 shows three year compound annual growth rate in sales (Panel A) and return on investment (ROI) (Panel B) for selected groups of companies. The first group has commercial banks. The second group has investment banks. There are no significant differences in the sales growths and ROIs of companies in these groups. Although Countrywide Financial Corp and Wachovia Corp didn’t survive the crisis, their fundamentals in terms of revenue growth and profitability are not different from the other banks. Similarly, from the investment banking group, Bear Sterns, Lehman Brothers and Merrill Lynch didn’t survive the crisis. But their revenue growth and ROI are not any worse than the other investment banks that survived. When we compare the retail groups, Linens N Things clearly performed worse than its competitor Bed Bath and Beyond. For example in 2004, Linens N Things compound annual sales growth rate was 13.43% compared to 20.7% for Bed Bath and Beyond. Similarly, the ROI of Linens N Things was only 7.5% compared to Bed Bath and Beyond’s 22.9% in 2004. Similar differences can be observed for the other years for which data are available. Similarly Best Buy’s sales growth and ROI significantly exceeded those of Circuit City Stores. In fact, Circuit City Stores incurred negative ROIs of -0.55% and -20.6% compared to Best Buy’s 20.17% and 27.32% for 2006 and 2007 respectively. Unlike the comparison of financial services firms, here we see clear differences in the fundamentals between the companies that survived the crisis (Bed Bath and Beyond and Best Buy) and those that didn’t survive (Linens N Things and Circuit City Stores). The bankruptcy of Linens N Things and Circuit City Stores is due to poor fundamental performances over the years. Companies with poor growth prospects and poor profitability cannot survive such severe downturns. Companies with good fundamentals can withstand adversities. Liquidity: refers to the ability of a company to quickly sell its assets and raise cash. Cash and short-term marketable securities are the most liquid of the assets. Companies that can sell their non-cash assets quickly when they need them to pay short-term obligations are considered liquid. Liquidity is important because if a company cannot pay its short-term obligations, the creditors can file for bankruptcy. During crisis, it is difficult for companies to sell stocks at reasonable prices to raise capital. Because investors lose confidence and stock prices decline. Many financial services firms either went bankrupt or were taken over by others at fire-sale prices due to poor liquidity. Bear Sterns was taken over by JP Morgan & Chase at $10 per www.intechopen.com Customer Challenges in Times of Global Risk and Uncertainty 53 share price10 on March 24, 2008. Only about a month before, Bear Sterns shares were trading above $80 per share. In August 2008, Lehman Brothers faced a similar problem. In spite of support by the Department of Treasury and the Federal Reserve, Lehman Brothers could not sell itself and in September, it filed for bankruptcy. In December 2008, it was Merrill Lynch’s turn, which sold itself to Bank of America. As we have seen in Table 2 above, these companies had sound fundamentals. The main cause of their collapse was lack of liquidity. They could not issue new securities to raise new capital, nobody wanted to deal with them because of lack of confidence, and unlike commercial banks, they could not have access to the Federal Reserve Discount Window Facilities. This indicates that the importance of liquidity cannot be overstated. Companies should maintain adequate cash and other liquid assets reserves for such eventualities. Fig. 4. Monthly closing prices of three investment banks that could not survive the crisis. Source: Based on price data obtained from Research Insight Figure 4 shows how fast the stock prices of the three investment banks fell due to lack of liquidity. The surviving investment banks such as Morgan Stanley and Goldman Sachs immediately applied for bank holding company status. The bank holding company status allows them access to the discount window facilities of the Federal Reserve Bank. A word of caution on the liquidity issue is that too much liquidity is unproductive. Cash and other liquid assets do not generate value by themselves. Value is created when the resources are invested in productive activities. Keeping large cash balances as reserve actually can result in a loss if inflation reduces the purchasing power of money. Therefore, The initial selling price agreed upon was $2.00 per share and the Federal Reserve Bank of New York supported the takeover. The offer price was increased later on to $10 per share to minimize the chance of shareholders’ opposition to the deal. 10 www.intechopen.com 54 The Economic Geography of Globalization appropriate balance should be maintained between the demand for liquidity and the need to invest available resources for productive purposes. Forecasting and Planning: the other precaution a company should make is proper forecasting and planning. Forecasting and planning don’t prohibit crisis from occurring, but they force advance thinking and preparation for possible eventualities. With forecasting and planning, the chance of surprises is reduced. Forecasts of many economic variables are available. Examples include gross domestic product, interest rates, government budget, exchange rates, etc. A company can relate its sales to these economic variables and estimate its future sales. Once sales forecast is determined, other financial aspects of the company can be projected. These projections will tell if the company needs to build up inventories, build facilities, raise more external capital, etc. The company should be prepared to act accordingly. The forecast should not be an estimation of a single figure. There should be scenario and what if analyses. These analyses help project possible deviations of economic conditions from the expected forecast and their possible consequences. Managers should make preparations accordingly. Strategic Adjustment: despite all the precautions and preparations, crises do occur. Crisis management involves visionary thinking and making strategic decisions. Such strategic decisions could be merger with other company, divestment of divisions, changing business model and legal status, etc. For example, during the 2007-2009 crises, Morgan Stanley and Goldman Sachs became bank holding companies. As investment banks, they could not accept demand deposits and access the Federal Reserve’s discount window facilities. The discount window facilities are available only to commercial banks. So conversion to bank holding company status allowed Morgan Stanley and Goldman Sachs to attract deposits and also access the discount window facilities. These allowed them to overcome their short-term liquidity problems while at the same time engaging in their core business of investment banking. Bear Sterns and Merrill Lynch merged with JP Morgan Chase and Bank of America respectively when they faced the same liquidity problem. Lehman Brothers could not do either and went bankrupt resulting in total loss to its stockholders. Citigroup sold some of its divisions and raised capital. The managers have to make such strategic decisions to assure survival and also increase shareholders’ wealth. 3.7 Summary The financial crisis of 2007-09 was triggered when real estate prices declined and mortgage defaults increased. Mortgage lenders were unable to recover the full balance of the loan due to decline in home prices. The effect is exacerbated because the mortgages have been securitized and floated in the markets and the resulting capital raised was loaned to finance more home purchases. The financial institutions were also undercapitalized due to a lapse in regulation. The repeal of the Glass-Steagall Act allowed banks to engage in securities activities and investment banks to engage in commercial banking. With the creation of special purpose entities, banks were able to remove mortgage backed securities off of their balance sheets and report in the balance sheet of such special purpose entities. This made it difficult for regulators to adequately enforce capital adequacy rules. With the crisis in the financial services sector, banks tightened credit to both households and businesses. This further reduced consumer confidence and discouraged spending, which caused spread of the crisis to all the economic sectors. Unemployment rate increased and exceeded 10%, many companies went bankrupt and stock markets lost about half of their values. www.intechopen.com Customer Challenges in Times of Global Risk and Uncertainty 55 The Department of the Treasury, the Federal Reserve Bank and the Federal Deposit Insurance Corporation made a coordinated effort to mitigate the impact of the crisis and speed up economic recovery. These government interventions took the form of outright lending of funds to troubled institutions, purchasing securities from these institutions, facilitating merger of weaker institutions with stronger ones, reducing interest rates and other general expansionary monetary policy to enhance liquidity and credit flow. The economy showed signs of recovery in the third quarter of 2009. The lessons that can be learned from the crisis are that companies with sound fundamentals and adequate liquidity have better chance of survival than those with poor fundamental and inadequate liquidity. In addition, prudent management requires making strategic adjustments in the form of mergers, changing business model, divestiture and other major decisions. Forecasting and planning with scenario and what-if analyses are also indispensable business management tools. 4. Global contextual issues and adaptive selling A key global marketplace solution for the volatile business challenges in today’s global economy has been seen in the concept of Adaptive Selling. Adaptive selling is defined as the salesperson’s ability to perform or “to take advantage of the unique communication elements associated with personal selling” (Weitz et al., 1986: 174). The latter, too, is important, according to those researchers: “Personal selling is the only [emphasis added] communication vehicle in which the marketing message can be adapted to the specific customer’s needs and beliefs” (174). The ideal concept of the practice of adaptive selling implies that the salesperson has the appropriate capabilities and sales conditions in place (Weitz et al., 1986). These ideal capabilities and conditions lead to the most effective of adaptive selling. At the extremes, a salesperson prepares a sales presentation for each customer (adaptive selling) or he/she uses the same presentation for all customers (adaptive selling’s opposite). The perceived information about the nature of the selling situation is the basis for the form of adaptive selling (Spiro and Weitz, 1990; Weitz et al., 1988). Extensive research has been conducted on the positive magnitude of the differences between adaptive selling and outcome performance and sales organization effectiveness measures (Babakus et al., 1996; Boorom et al., 1996; Piercy et al., 1999; Sujan et al., 1994). Little, however, has been written about the contextual issues of adaptive selling. This part of the chapter aims, then, to contribute to that aspect of the topic, and is divided into three main sections. The first presents a framework for examining adaptive selling and contextual issues. Prior research on contextual issues, and how they are related to adaptive selling, is discussed in the second section. This part of the chapter concludes with a discussion of contextual variables that support salespeople in practicing adaptive selling and learning from their experiences. 4.1 A framework of contextual issues and adaptive selling The model for “An adaptive Selling Framework” from Weitz et al. (1986) includes environmental conditions that we relate to global contemporary contextual issues to the practice of adaptive selling. This model identifies some key aspects associated with environmental conditions and adaptive selling and suggests the ways in which these are interrelated. It is not intended to describe the variables and processes of the practice of adaptive selling itself. The model of adaptive selling focuses on the behavior of the www.intechopen.com 56 The Economic Geography of Globalization salesperson and is influenced by the characteristics of salesperson and sales management variables (Weitz et al., 1986). This model is consistent with recognized research paradigms (Walker et al., 1979; Weitz, 1981; Weitz et al., 1986; Baldauf & Cravens, 2002) and uses moderators represented by salesperson capabilities (which can be separated, for example, into selling skills, product knowledge, and information collection), and motivational (intrinsic reward orientation and strategic analysis), organizational (type of product), and environmental (industry growth) differences. This section relies on previous research surrounding the relationship between the practices of adaptive selling and the behavior of the salesperson moderated by the environmental conditions in which the sale takes place. Weitz et al. (1986) suggest three main characteristics of the selling environment that influence the outcome of adaptive selling: “(1) the variety of customer needs and type encountered by the salesperson, (2) importance of the typical buying situation encountered, and (3) the resources provided by the company to the salesperson” (176). In this section, similar environmental conditions are examined, but within contextual issues extracted from theories of contextualization. Contextualization theories include the contributions of the Chicago School of Sociology (Barley, 1989), the social theory of Pierre Bordieu (1977), the structuration theory of Anthony Giddens (1984), and others. The main issues from previous contextual theories selected as relevant to adaptive selling are the context of work, the context of origin, the context of society and culture, and the global context. Proposition 1: The practice of adaptive selling is moderated by the characteristics of the selling environment and by the larger context of work, in the context of origin, in the context of society and culture, and in the global context. Focusing on salesperson behavior, as suggested by Weitz et al. (1986), the variety of customer needs, the typical buying situation, and the resources provided by the company are interrelated with the context of work, the context of society and culture, and the global context. These issues are discussed in greater detail with other propositions in the next section. 4.2 The context of work needed to practice adaptive selling In the world of working and organizing, substantial changes have been observed over the past decades. The practice of adaptive selling faces issues such as new forms of working and organizing and work-related social relationships. There is no global uniformity, and countries differ to a considerable degree in the flexibility of labor markets, with a tendency toward deregulation of national employment systems, and increasing importance placed on global markets (Dore, 2004). New forms of working and organizing have been an important theme in management research (Ruigrok et al., 1999; Whittington, et al. 1999) as well as for political decision makers over the past decade (Savage, 2001). With the changing organizational environment that is constituted by new information technologies (Gattiker & Coe, 1986), there are new ways of working, which include workers holding multiple jobs, enduring precarious working arrangements, and undergoing frequent occupational changes. Proposition 2: New contextual forms of working are critical to adaptive selling. When confronting a sales situation that involves customers holding multiple jobs, working under precarious labor contracts, or undergoing frequent occupational changes, adaptive salespeople tend to make an effort to integrate and follow customers on the basis of their working arrangements. www.intechopen.com Customer Challenges in Times of Global Risk and Uncertainty 57 The variety of customer needs grows with higher uncertainty in housing, income, and relationships. They may not buy the same products as before, for example expensive furniture items, since they must move frequently, enjoy lower disposable incomes, or prefer to invest in other types of assets (such as reliable financial products). Company resources for the salesperson targeting customers in the new working conditions of today differ significantly. Since these customers seldom place large orders and do without large investment-information-based purchases, the benefits for the adaptive salesperson may not be substantial. Companies may adopt, for example, self-service or web-based means of selling their products and services, reducing adaptive selling related to salesperson behavior. The social environment is another important issue for adaptive selling, since individuals can mirror themselves in the larger social context. Estimations about one’s relative position in a social context are not developed autonomously. Social comparisons are influenced by the social identity of individuals. The image the social environment holds about individuals contributes to that with which they are entrusted, which development offers they receive, and how they are evaluated. Proposition 3: The relative position of the salesperson in a social context is critical for adaptive selling. When the salesperson confronts the image the social environment holds about him/her, it contributes to that with which he/she is entrusted in sales. Proposition 4: The relative position of individuals in a social context is critical for adaptive selling. When an individual desires to make a difference in the image the social environment holds of him/her, the adaptive salesperson can customize and tailor to the needs of his/her customers. The first proposition relates to company resources. The image customers hold about the salesperson is important for the practice of adaptive selling. If the salesperson has no credibility in the applicable social environment, he/she may be precluded from advancing his/her sales practice. This situation may come about for several reasons, for example, if the salesperson has been involved in a publicized litigation issue. Under this circumstance, the company may withhold the resources the salesperson needs to conduct adaptive selling. The second proposition is highly inspiring for adaptive salespeople. If individuals in the environment in which the sales are practiced consider individual customization an important feature to differentiate their social images, the salesperson will come to the typical buying situation with a variety of tools (company resources) to meet those customer needs. Other important contextual issues within the context of work are networking and mentoring both outside and within organizations. Networking is the process of building up and maintaining a set of informal, cooperative relationships in the social structure of an organization (Burt, 1992). Networks provide opportunities. They offer contacts and supporters that increase positive outcomes in negotiations and the number of options and choices available. The issue of mentoring is linked with the topic of networking. It is a particular kind of interpersonal relationship in which protégés receive a broad range of job and psychological help from senior managers (Kram, 1988). It has been connected with training and the development of capabilities (Hunt & Michael, 1983). Proposition 5: Networking is important for salespeople practicing adaptive selling. Increasing contacts and supporters (interest on the product or service) is crucial since it engenders a greater variety of customer options and choices. Proposition 6: If the salesperson is mentored by a senior colleague, the salesperson’s capabilities for adaptive selling in identifying contextual work situations are facilitated. www.intechopen.com 58 The Economic Geography of Globalization These two propositions regard company resources provided to enhance the practice of adaptive selling. The salesperson can identify the ideal contextual work conditions regarding company resources. 4.3 The context of origin Regarding society and culture, four major aspects can be said to constitute the important contextual elements for adaptive selling: gender, ethnicity, demography, and communal and societal ties. When authors discuss gender as a contextual variable, they tend to use it as a control variable (see Turban & Dougherty, 1994) or in line with change reflecting societal conditions that provide opportunity structures (see Fielden & Davindson, 2010). For adaptive selling, gender income differences and the participation of males and females in the labor market are important. Proposition 7: The context of origin needed to practice adaptive selling needs to take into account gender income differences. Identifying gender income differences allows the salesperson to identify the respective purchase power of males and females as well as their needs. Ethnicity concerns the question of discrimination based on race or membership in an ethnic minority group. A reduction of opportunities exists for ethic others within a homogenous population. Homogeneousness is the degree of demographic and identity similarity of interacting individuals (Ibarra, 1993). Proposition 8: The context of origin needed to practice adaptive selling needs to take into account how individuals interact in the context wherein the sales are practiced. Identification of the importance of contextual ethnicity informs the salesperson about the typical buying situation and whether within ethic groups there are different customer needs. If the salesperson identifies with a certain ethic group, the practice of adaptive selling is facilitated. Often, demographics are related to world regions, nation states, or occupations, and serve as a point of reference for many disciplines. Regarding adaptive selling, the composition of corporate elites (Stanwroth & Giddens, 1974), and the perception and consequences of age (Lawrence, 1988) may be important contextual aspects of origin. Proposition 9: The context of origin needed to practice adaptive selling involves analyzing the composition of social elites. Proposition 10: The context of origin needed to practice adaptive selling involves analyzing the perception and consequences of age. The context of origin regarding demographic fluctuations and perceptions of social elites and age is important if the salesperson is to identify the purchasing power and needs of local customers. For example, if the local population is elderly and elitist, it may be the case it has unique and expensive tastes in products and services. The role of community is another important context of origin since it concerns the integration of individuals into the local context of civil, political, and religious cooperation. Proposition 11: The context of origin needed to practice adaptive selling needs to take into account how individuals are integrated in the local civil, political, and religious communities wherein the sales are practiced. The context of origin, regarding the ways in which individuals are integrated in local civil, political, and religious communities, facilitates adaptive selling in recognizing the typical www.intechopen.com Customer Challenges in Times of Global Risk and Uncertainty 59 buying situations and variety of customers. For example, if the local community holds strong political views about the certain country wherein a product is manufactured, its members may refuse to buy the product. The same can be said for religious views, for example, regarding food, or community civil views, for example, regarding sexual services. 4.4 The global context Due to the increasing amount of business conducted at an international level, individuals and companies work hard to try to access international labor markets (Vance, 2002). Virtualization is one of the societal developments to have received considerable attention mainly within the study of virtual teams (Jong et al., 2008). These virtual interactions go beyond frequent commuting, continuous short-term visits, or enhanced communication opportunities such as video conferencing (Mayerhofer, Hartmann, Michelitsch-Riedl, & Kollinger, 2004). Virtualization practices in organizations increase the interpretive complexity of contextualization conventions (Von Glinow et al., 2004). Moreover, people tend to interpret the meaning of virtual procedures according to their contextual knowledge or to what is local to them (Von Glinow et al., 2004). In virtual management, conventions subsist and the awareness of the other side’s networks of relationships and their interpretations may be unknown. Since virtual management provides no contextual meaning of informality, assumptions about others and their contexts are drawn, which causes constraints in the flow of business. Proposition 12: New contextual forms of work-related social relationships are critical for adaptive selling. When confronting a sales situation that involves networking with virtual relationships, adaptive salespeople tend to assume the context of the sales interaction. The global context regarding virtualization implies changes in the ways traditional adaptive selling has been recognizing typical buying situations and the varieties of customers that exist worldwide. 4.5 Summary Personal adaptive selling is an active process that can be facilitated or hindered by contextual conditions. This section discussed the customer contexts of work, origins, society and culture, and virtual interactions in the global context. Most studies to date have focused on the relationship between salesperson behavior and performance and outcomes in sales for organizations, including the research on moderators such as capabilities of the salesperson, type of industry growth, and type of product (see Baldauf and Cravens, 2002). However, contextual issues have been largely ignored. This section provides a means for the development of measures of the key constructs to test propositions. These measures must be validated in the area of the personal adaptive selling domain. An obvious way to measure the practice of adaptive selling is by assessing the degree to which salespeople vary their behaviors across contexts, including the variance of contextual selling situations encountered by the salesperson. The salesperson’s attention to contexts and his/her capability to recognize typical situations and customer variety allows for the forging of appropriate adaptive selling strategies. This section suggests a salesperson acts as a “chameleon” by modifying and controlling sales presentations. Sales may not be facilitated in all contexts, particularly those where issues of www.intechopen.com 60 The Economic Geography of Globalization origin (such as ethnicity and gender) may be at stake, unless the salesperson has a strong identity relating to such groups. The proposed framework suggests methods for developing contextual structures and guides for salespeople in identifying important issues in adaptive selling. It enables salespeople to exploit unique opportunities for sales facilitated by contextual influence as well as situations wherein their interpersonal influence may be difficult to apply. These future directional contextual aspects warrant further research attention because they are not typically related to the development and utilization of the skills needed to operate effectively, but rather to how the capabilities can be easily used or hindered in different contexts. These propositions represent a new direction in the practice of adaptive selling. In outlining the testable propositions, some evidence was provided, largely drawn from domains other than personal selling. Thus, it is necessary to be prudent in interpreting the propositions until they are tested in the adaptive selling domain. Most of these propositions can be tested with survey methods, whereas others are open to experimental design. It was not the aim of this section to propose effective sales approaches, but rather the ways in which contexts can support salesperson’s capabilities in and motivation for adaptive selling. 5. References Babakus, E., Cravens, D.W., Grant, K., Ingram, T.N. and LaForge, R.W. (1996), Investigating the relationship among sales management control, sales territory design, salesperson performance, and sale organization effectiveness, International Journal of Research in Marketing, Vol. 13, pp.345-63 Baldauf, A. and Cravens, D.W. (2002), The effect of moderators on the salesperson behavior performance and salesperson outcome performance and sales organization effectiveness relationships, European Journal of Marketing, Vol.36 No.11/12, pp. 1367-1388 Barley, S. R. (1989), Careers, identities, and institutions: The legacy of the Chicago school of sociology. In M.B. Arthur, D. T. Hall, and B.S. 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(1999), Change and complementarities in the new competitive landscape: A European panel study, Organization Science, Vol. 10 No. 5, pp. 583-600 www.intechopen.com The Economic Geography of Globalization Edited by Prof. Piotr Pachura ISBN 978-953-307-502-0 Hard cover, 264 pages Publisher InTech Published online 27, July, 2011 Published in print edition July, 2011 Very often the process of globalization is referred the word economy evolution. Often we measure and study globalization in the economic relevance. The economy is possibly the most recognized dimension of globalization. That is why we see many new phenomena and processes on economic macro levels and economic sectoral horizons as well as on specific “geography of globalizationâ€​. The book The Economic Geography of Globalization consists of 13 chapters divided into two sections: Globalization and Macro Process and Globalization and Sectoral Process. The Authors of respective chapters represent the great diversity of disciplines and methodological approaches as well as a variety of academic culture. This book is a valuable contribution and it will certainly be appreciated by a global community of scholars. How to reference In order to correctly reference this scholarly work, feel free to copy and paste the following: Demissew Ejara, Christina Reis, Walter Carter and Tony Carter (2011). Customer Challenges in Times of Global Risk and Uncertainty, The Economic Geography of Globalization, Prof. Piotr Pachura (Ed.), ISBN: 978953-307-502-0, InTech, Available from: http://www.intechopen.com/books/the-economic-geography-ofglobalization/customer-challenges-in-times-of-global-risk-and-uncertainty InTech Europe InTech China University Campus STeP Ri Slavka Krautzeka 83/A 51000 Rijeka, Croatia Phone: +385 (51) 770 447 Fax: +385 (51) 686 166 www.intechopen.com Unit 405, Office Block, Hotel Equatorial Shanghai No.65, Yan An Road (West), Shanghai, 200040, China Phone: +86-21-62489820 Fax: +86-21-62489821 © 2011 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution-NonCommercialShareAlike-3.0 License, which permits use, distribution and reproduction for non-commercial purposes, provided the original is properly cited and derivative works building on this content are distributed under the same license. 

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doi:10.14720/aas.2022.118.3.2419 Review article / pregledni znanstveni članek Razvoj raziskovalnih metod za karakterizacijo združb arbuskularnih mikoriznih gliv in potencialni vpliv biodiverzitete glivnih endofitov na vegetacijo Irena MAČEK 1, 2 Received November 12, 2021; accepted July 13, 2022. Delo je prispelo 12. novembra 2021, sprejeto 13. julija 2022 Development of research methods to characterise arbuscular mycorrhizal fungal communities and potential effects of fungal endophyte biodiversity on vegetation Abstract: Characterization and quantification of the functional and taxonomic diversity of microbial communities is essential for understanding all aspects of microbial ecology and is closely related to ecosystem function. Arbuscular mycorrhiza is the most widespread symbiosis on Earth, with arbuscular mycorrhizal (AM) fungi present in more than 2/3 of all plant species. Just over a decade after the publication of the first review article on molecular approaches to study the ecology of AM fungi in Acta Agriculturae Slovenica (Maček, 2009), the rapid development of molecular tools, especially next generation sequencing (NGS) technology, has accelerated the study of the research field of plant root endophytes. In this paper, the current approach to study the ecology and taxonomy of AM fungi is presented, which also provides some insights into the study of other plant root endophytes. In addition, a widely used system for classifying AM fungi with so-called virtual taxa (VT) is presented, which is used for ecological studies and comparison between different studies. Finally, a brief overview of the importance of climate and soil properties for AM fungal community composition and taxa distribution in global ecosystems is presented. Key words: arbuscular mycorrhiza; biodiversity; ecology; endophytes; rhizosphere; sequencing; soil Razvoj raziskovalnih metod za karakterizacijo združb arbuskularnih mikoriznih gliv in potencialni vpliv biodiverzitete glivnih endofitov na vegetacijo Izvleček: Karakterizacija in kvantifikacija funkcionalne in taksonomske raznolikosti mikrobnih združb je ključnega pomena za razumevanje vseh vidikov mikrobne ekologije in je povezana tudi širše z razumevanjem delovanja ekosistemov. Arbuskularna mikoriza predstavlja najbolj razširjeno in starodavno simbiozo na Zemlji, saj so arbuskularne mikorizne (AM) glive prisotne v koreninah več kot dveh tretjin vseh rastlinskih vrst. V dobrem desetletju od objave preglednega članka o uporabi molekulskih pristopov pri raziskavah arbuskularne mikorize v reviji Acta Agriculturae Slovenica (Maček, 2009) je razvoj metodologije, predvsem tehnologije določanja nukleotidnega zaporedja (sekvenciranja) naslednjih generacij (NGS), močno pospešil raziskave raznolikosti in ekologije združb AM gliv in drugih koreninskih endofitov. V tem članku so predstavljene novosti na področju raziskav endofitskih gliv v koreninah rastlin, s poudarkom na aktualnem pristopu k raziskavam v ekologiji in taksonomiji AM gliv, ter sistem njihove klasifikacije s tako imenovanimi virtualnimi taksoni (VT). Slednji je zelo uporaben za namen ekoloških raziskav in širše primerjave različnih študij med sabo. Na kratko je predstavljen tudi vpliv klimatskih in talnih lastnosti okolja na sestavo združb in pojavljanje posameznih taksonov AM gliv v različnih ekosistemih. Ključne besede: arbuskularna mikoriza; biodiverziteta; ekologija; endofiti; rizosfera; sekvenciranje; tla 1 Oddelek za biologijo, Biotehniška fakulteta, Univerza v Ljubljani, Ljubljana, Slovenija 2 Korespondenčni avtor, e-naslov: irena.macek@bf.uni-lj.si Acta agriculturae Slovenica, 118/3, 1–13, Ljubljana 2022 I. MAČEK 1 UVOD V RAZISKAVE DIVERZITETE AM IN DRUGIH SKUPIN ENDOFITSKIH GLIV V KORENINAH RASTLIN Arbuskularne mikorizne (AM) glive (skupina Glomeromycotina ali tudi Glomeromycota) (Spatafora in sod., 2016, Tedersoo in sod., 2018) predstavljajo ključno skupino talnih mikroorganizmov v številnih kopenskih ekosistemih in najbolj široko razširjeno simbiozo na planetu (Brachmann in sod., 2006). Povezava med AM glivami in rastlinami je starodavna, saj so bile AM glive prisotne že ob prehodu rastlin iz morja na kopno v paleozoiku pred več kot 450 milijoni let. Poleg AM gliv (Glomeromycotina) pa so bile ob prehodu rastlin na kopno prisotne tudi endofitske glive iz starodavne in delno saprotrofne skupine Endogonales (Mucoromycotina, Mucoromycota), katerih predstavnike so dolgo uvrščali med AM glive zaradi podobnih morfoloških struktur (drobne hife in njihov razrastki podobni abuskulom), ki jih te glive tvorijo v koreninah rastlin. Na podlagi molekulskih označevalcev (markerjev) je bilo ugotovljeno, da predstavljajo t.i. drobni koreninski endofiti iz skupine Mucoromycotina (ang. ‚fine root endophytes‘ ali MFRE) filogenetsko ločeno skupino globalno razširjenih rastlinskih endosimbiontov (Orchard in sod. 2017). Slednji tudi danes še vedno tvorijo endosimbiozo z večino skupin kopenskih rastlin, pogosto tudi istočasno in funkcionalno komplementarno z AM glivami (Field on sod., 2015; Orchard in sod., 2017, Hoysted in sod., 2019, Besiana et al., 2021; Sinanaj in sod., 2021). Ker so ugotovitve o pomenu simbioze rastlin z glivami iz skupine Mucoromycotina še relativno nove, je podatkov o njihovi povezavi z rastlinami manj kot za arbuskularno mikorizo, tako da obstaja še kar nekaj odprtih vprašanj za boljše razumevanje te skupine gliv in njihove funkcije v ekosistemih (Sinanaj in sod., 2021). V tem preglednem članku se osredotočam predvsem na arbuskularno mikorizo, torej AM glive iz skupine Glomeromycotina, vsekakor pa bo v prihodnosti potrebno spremljati tudi razvoj raziskav drugih skupin rastlinskih endofitov, tako že dlje časa poznanih temnih septiranih endofitov (DSE) (Rodriguez in sod., 2009, Knapp in sod., 2018, Tonjer in sod., 2021), kot tudi drobnih koreninskih endofitov iz skupine Mucoromycotina (MFRE). Razvoj novih molekulskih metod v zadnjem desetletju vsekakor omogoča lažje odkrivanje in razumevanje celotnega spektra raznolikosti organizmov, ki živijo v območju rastlinskih korenin oz. rizosferi. V bližnji prihodnosti zato lahko pričakujemo veliko novih informacij o starodavnih simbiozah med glivami in rastlinami, njihovi biodiverziteti in pomenu za ekosisteme, vključno z agroekosistemi. Že dolgo je znano, da arbuskularna mikoriza rastlinam prinaša številne koristi, od izboljšane mineralne 2 Acta agriculturae Slovenica, 118/3 – 2022 prehrane in preskrbe z vodo ter obrambe pred patogeni, do posrednih koristi npr. izboljšane strukture tal in manjše erozije tal. AM glive so obvezni biotrofi, od rastlin dobivajo fotoasimilate, ki predstavljajo njihov edini vir organskega ogljika (Smith & Read, 2008). Simbioza vpliva tudi na sestavo združb kopenskih rastlin, s tem pa tudi na funkcioniranje ekosistemov in njihovo produktivnost (Fitter, 2005, Schnitzer & Klironomos, 2011, Wurzburger in sod., 2017). Taksonomsko lahko AM glive klasificiramo na več načinov, od tradicionalnih klasifikacij, ki temeljijo na morfologiji celične stene njihovih spor (npr. Oehl in sod., 2011, Blszkowski, 2012), kar je naslovljeno v prvem delu članka, do molekulskih pristopov, pri katerih uporabljamo različne molekulske označevalce, kar je obravnavano v drugem delu članka. Definicija vrste je pri mikroorganizmih, vključno z mikroglivami, na splošno zelo težavna in podvržena konsenzu na podlagi trenutnega poznavanja posamezne skupine organizmov. Prav zato lahko na tem področju v prihodnosti pričakujemo tudi nadaljnje spremembe klasifikacije, ki bodo temeljile na novo pridobljenih podatkih o AM glivah. V zadnjem delu članka so na kratko povzete tudi glavne omejitve različnih pristopov k raziskovanju raznolikosti AM gliv, s poudarki, kje je na mestu previdnost, z namenom čim bolj kvalitetne interpretacije rezultatov in pridobivanja novega znanja o tej zanimivi in pomembni skupini organizmov. 1.1 GOJENJE AM GLIV V ČISTIH KULTURAH IN KLASIČNA IDENTIFIKACIJA VRST AM GLIV Veliko težavo pri morfološkem določanju vrst AM gliv predstavlja njihovo gojenje v čistih kulturah, saj AM glive brez primerne gostiteljske rastline ne uspevajo. V sklopu raziskav AM gliv čisto, enovrstno (monospecifično) kulturo predstavlja kultura, kjer je prisotna samo ena vrsta glive, ki je bila vzgojena iz ene same spore. Postopek vzpostavitve take kulture je zapleten in dolgotrajen, zahteva veliko ekspertnega znanja ter rastne razmere, ki preprečujejo navzkrižno kontaminacijo lončnih kultur rastlin z drugimi glivami iz okolja (npr. ob zalivanju rastlin, zaradi prenosa z živalskimi vektorji, kot so insekti itd.). Spore nekaterih vrst AM gliv lahko izoliramo iz njihovega okolja (običajno iz talnih vzorcev ali pri nekaterih vrstah tudi iz korenin) s postopkom redčenja in koncentriranja v vodi in/ali raztopini saharoze ter iskanjem in prepoznavanjem spor s stereo mikroskopom. Novo, enovrstno lončno kulturo AM gliv vzpostavimo s prenosom posamezne spore v bližino korenin izbranih vrst rastlin. Rastline so običajno za namen tega postopka vzgojene iz semen, ki so bila predhodno površinsko sterilizirana. Da povečamo možnost neposrednega stika Razvoj raziskovalnih metod za karakterizacijo združb arbuskularnih mikoriznih gliv in potencialni vpliv ... na vegetacijo Slika 1: Vzpostavitev enovrstnih (monospecifičnih) kultur AM gliv, vzgojenih iz ene same spore, v nastavkih za pipete (levo) in kasneje v lončnih kulturah (sredina, desno). Namen tega postopka je, da omogočimo čim boljši stik korenine rastline s posamezno izolirano sporo AM gliv in kasneje pomnoževanje AM gliv v kulturi. Rastline so vzgojene iz semena, ki je bilo predhodno površinsko sterilizirano (foto: I. Maček) Figure 1: Monospecific cultures of AM fungi grown from a single AM fungal spore in pipette tips (left) and later in pot cultures (centre, right). The method allows good spatial contact between plant roots grown from a single surface-sterilised seed and a single AM fungal spore and further fungal development (photo: I. Maček) med korenino rastline in viabilno sporo AM glive, vzpostavimo začetno interakcijo med obema organizmoma v omejenem prostoru. Za ta namen se je izkazala primerna uporaba nastavkov za pipete, napolnjenih z vlažnim, sterilnim substratom (Slika 1). Kasneje rastline skupaj z nastavkom prenesemo v večje lonce, napolnjene s sterilnim substratom (Slika 1). Da zagotovimo bolj raznolik življenjski prostor za glive (koreninska biomasa) lahko v kasnejši fazi rasti dosejemo še več gostiteljskih rastlin. Koristno je, če uporabimo več rastlinskih vrst, kar omogoči večji nabor potencialnih gostiteljev za AM glive, večjo raznolikost in biomaso korenin in s tem večjo možnost, da se simbioza (mikoriza) v rizosferi dejansko vzpostavi. Pomembno je, da tudi kasneje, tekom rasti rastlin, maksimalno zmanjšamo možnost navzkrižne kontaminacije s sporami ali delci infektivnih hif med posameznimi lončnimi kulturami. Taki preventivni postopki zajemajo gojenje rastlin v zaprtih prostorih, kontrolo škodljivcev, kontrolirano odtekanje vode iz loncev z mikorizo, ustrezno zalivanje, ki je lahko urejeno z avtomatiziranim kapljičnim sistemom, in omejeno ventilacijo zraka znotraj prostora. Rastline lahko gojimo tudi v posebnih vrečkah z vgrajenim filtrom, kjer se ustvari mikroklima in so izolirane od ostalih rastlin, obenem pa je poseganje v tak sistem (npr. zalivanje) omejeno na minimum. Vzpostavitev enovrstne kulture AM gliv je dolgotrajni postopek, ki je lahko odvisen od številnih de- javnikov, med drugimi vrste oz. genotipa glive, rastne sezone, gostiteljskih rastlin in rastnih razmer. Tudi sporulacija (tvorba spor) je pri AM glivah zelo nepredvidljiva in odvisna od vrste glive. Namen vseh teh postopkov je zagotovitev zadostne količine materiala (nepoškodovanih, živih spor) za njihovo izolacijo in identifikacijo, saj za opis morfološke vrste AM gliv ne zadostuje ena sama spora, ampak jih potrebujemo vsaj nekaj deset za pripravo preparatov in še več za kasnejše shranjevanje glivnega materiala v banko gliv (Oehl in sod., 2011), kar je pogoj za opis nove vrste (Slika 2). Običajno se shranjuje posušen substrat s sporami v suhem, temnem in hladnem prostoru oz. se vzdržuje aktivno kulturo AM gliv, skupaj z živimi rastlinskimi simbionti v rastlinjaku, kar pa je časovno in finančno zelo zahtevno. Taksonomske in ekološke raziskave AM gliv na podlagi morfološh znakov so torej zelo težavne iz več razlogov. Če povzamemo, so glavne omejitve: (1) veliko taksonov AM gliv ne moremo gojiti v čistih (lončnih) kulturah, obenem je glive nemogoče identificirati na podlagi kolonizacije v koreninah (Slika 3), (2) zahteve za rast in sporulacijo različnih taksonov AM gliv so zelo raznolike in kompleksne, (3) pri številnih taksonih (še) nismo uspeli izolirati njihovih spor in tako morfološko določene vrste povezati s podatki, ki izvirajo iz molekulskih raziskav na osnovi DNA, (4) taksonomsko določanje AM gliv je zelo zapleteno in zahteva kompleksno ekspertno znanje, ki je Acta agriculturae Slovenica, 118/3 – 2022 3 I. MAČEK Slika 2: Spore dveh različnih vrst AM gliv iz rodu Rhizophagus (levo, sredina). Spora AM glive s strto celično steno iz rodu Acaulospora (desno). Vidna je večplastna, strukturirana celična stena. Morfološke značilnosti celične stene, njenih plasti ter pritrjenih hif služijo kot taksonomski znaki v taksonomiji AM gliv (foto: I. Maček) Figure 2: Spores of two different AM fungal species from the genus Rhizophagus (left, centre). Crushed AM fungal spore of the genus Acaulospora with visible multi-layered structured cell wall (right). The morphological structures of the cell wall, its layers and attached hyphae serve as taxonomic features in the taxonomy of AM fungi (photo: I. Maček) kljub temu, da na to temo obstaja kar nekaj strokovne literature (Oehl in sod., 2011, Blaszkowski, 2012), omejeno na samo nekaj strokovnjakov (Slika 2), (5) taksonomski znaki različnih skupin AM gliv se lahko tudi prekrivajo, oz. ni znano, če lahko npr. ista gliva v različnih okoljih tvori tudi več različnih morfoloških tipov spor. Prav zato je razvoj molekulskih metod zelo pomemben za razumevanje ekologije in diverzitete AM in drugih endofitskih gliv. Ena izmed prednosti tega pristopa je tudi ta, da lahko DNA izoliramo direktno iz korenin (Slika 3), s tem pa dobimo podatke o združbi gliv, ki je fiziološko aktivna in v času vzorčenja v dejanski interakciji z rastlinami. Molekulske metode in njihov hitri razvoj tako predstavljajo pravo revolucijo v razumevanju teh organizmov in so še vedno med najbolj obetavnimi orodji za raziskave združb endofitskih gliv (Clapp in sod., 2003, Dickie & FitzJohn, 2007, Dumbrell in sod., 2017, Sinanaj in sod., 2021). 2 MOLEKULSKA IDENTIFIKACIJA AM GLIV V OKOLJSKIH VZORCIH IN GENSKI MARKERJI Dejstvo je, da obstaja relativno malo vrst oz. taksonov AM gliv, ki jih lahko gojimo v lončnih kulturah. Pri številnih genotipih AM gliv, ki jih poznamo samo na osnovi molekulskih označevalcev izolirane DNA iz 4 Acta agriculturae Slovenica, 118/3 – 2022 okoljskih študij, raziskovalci še nismo uspeli izolirati spor ali celo ugotoviti, če ti taksoni sploh sporulirajo v njihovem naravnem okolju. Taki genotipi so poznani samo na osnovi nukleotidnih zaporedij oz. sekvenc določenih genskih označevalcev. V okoljskih študijah se kot ustrezni označevalec za raziskave raznolikosti in ekologije združb AM gliv največkrat uporablja gen 18S rRNA za malo podenoto ribosoma (SSU) (Simon in sod., 1992, Helgason in sod., 1998; Maček in sod., 2019), v zadnjem času pa v manjšem obsegu tudi regija notranjega prepisanega vmesnika – ITS (nuclear ribosomal internal transcribed spacer region) (npr. Alzarhani in sod., 2019) (Slika 4). Slednja se največkrat uporablja v primeru, da so v raziskavo vključene tudi druge skupine gliv (ne samo AM glive), za katere je regija ITS bolj primeren označevalec kot 18S rRNA, obenem pa uporaba ene same regije za vse zmanjša stroške raziskave (uporaba enega označevalca, namesto dveh, čeprav regija ITS ni optimalno za identifikacijo AM gliv) (Alzarhani in sod., 2019). Molekulsko določanje omogoča številčno ovrednotenje (kvantifikacijo) taksonov AM gliv v tleh ali v koreninah rastlin. Geni, ki kodirajo to 18S rRNA (ali 18S SSU) genomsko regijo, so dostopni v velikem številu kopij in vsebujejo veliko ohranjenih kot tudi variabilnih regij, kar omogoča ločevanje taksonov na različnih ravneh. Sekvence male podenote ribosoma (18S SSU) se v ekologiji AM gliv uporabljajo nekje od začetka devetdesetih let prejšnjega stoletja (Simon in sod., 1992). Do Razvoj raziskovalnih metod za karakterizacijo združb arbuskularnih mikoriznih gliv in potencialni vpliv ... na vegetacijo Slika 3: Kolonizacija korenin koruze (Zea mays L.) z AM glivami (levo) in mikroskopski preparati za oceno stopnje kolonizacije korenin z AM glivami (desno). Na sliki levo so vidne znotraj-koreninske hife AM gliv in arbuskuli (drevescom-podobne strukture), ki so pomembne za izmenjavo hranil med rastlinami in glivami (foto: I. Maček) Figure 3:. Arbuscular mycorrhizal fungi colonising maize (Zea mays L.) roots (left), with visible intraradical hyphae and arbuscules (tree-like structures) important for nutrient exchange between plants and fungi. Slides for microscopy and estimation of AM fungal colonisation in plant roots (right) (photo: I. Maček) danes je bilo objavljenih kar nekaj različnih začetnih oligonukleotidov za pomnoževanje odsekov sekvenc, specifičnih za različne skupine AM gliv, v postopku verižne reakcije s polimerazo (PCR). Najpogosteje uporabljen par začetnih oligonukleotidov male podenote ribosoma v okoljskih raziskavah je AM1 (Helgason in sod., 1998) in NS31 (Simon in sod., 1992), kjer je dolžina pomnoženega fragmenta DNA približno 550 baznih parov. Za ta par začetnih oligonukleotidov je značilno tudi za AM glive nespecifično pomnoževanje drugih fragmentov, sploh, kadar je glivne DNA v okolju oz. ekstraktu malo. To je bil tudi povod za izdelavo novih začetnih oligonukleoti- dov, kot je par AML1-AML2 (Lee in sod., 2008; dolžina pomnoženega fragmenta DNA je okrog 800 baznih parov) (Slika 4). Regija vsebuje tudi začetno regijo pomnoževanja oligonukleotidov AM1-NS31, in pomnožuje širok nabor taksonomskih skupin AM gliv, ne pa vseh – izključujeta na primer skupino Paraglomeraceae (Lee in sod., 2008). Ribosomska DNA je v posamezni spori AM gliv zelo polimorfna, kar pomeni, da so sekvence rRNA gena med posameznimi taksoni AM gliv močno variabilne (Tisserant in sod., 2013). Dokazano je tudi, da hife in spore AM gliv vsebujejo na stotine jeder (Tisserant in Slika 4: Shematski pregled najpogosteje uporabljenih parov začetnih oligonukleotidov, ki se uporabljajo v ekoloških raziskavah AM gliv (NS31-AM1 in AML1-AML2). Na vrhu slike so prikazani molekulski označevalci jedrne ribosomske RNA – mala podenota ribosoma (18S SSU), velika podenota ribosoma (28S LSU), 5.8S medgenski vmesnik (IGS) in notranji prepisani vmesnik (ITS). Trikotne puščice prikazujejo smer in mesto za prijemanje začetnih oligonukleotidov Figure 4: Schematic of the most common primer pairs and DNA regions used in the community ecology of AM fungi (NS31-AM1 and AML1-AML2). At the top of the figure are shown molecular markers of nuclear ribosomal RNA (rRNA) – small ribosomal subunit (18S SSU), large ribosomal subunit (28S LSU), 5.8S intergenic spacer (IGS) and the internal transcribed spacer (ITS). The arrows indicate the direction and alignment range of the primers Acta agriculturae Slovenica, 118/3 – 2022 5 I. MAČEK sod., 2013). Zaradi polimorfizma trenutno razpoložljivih genskih označevalcev je težko določiti vrsto AM gliv z molekulskimi metodami. V večini študij združb AM gliv različne taksone identificiramo kot skupine sorodnih sekvenc (npr. operativne taksonomske enote – OTU), ki pa verjetno bolj kot nivoju posamezne vrste AM gliv ustrezajo posameznim rodovom (npr. Krüger in sod., 2009). Največ okoljskih raziskav raznolikosti AM gliv je bilo izvedenih s pomnoževanjem regije 18S rRNA (npr. Schwarzott & Schuβler, 2001; Helgason in sod., 2002; Vandenkoornhuyse in sod., 2002; Öpik in sod., 2006), najprej z uporabo pirosekvenciranja s platformo Roche 454 GS-FLX (npr. Öpik in sod., 2009, Dumbrell in sod., 2011), kasneje pa tudi s z določanjem nukleotidnega zaporedja (sekvenciranjem) s platformo Illumina (npr. Alzarhani in sod. 2019; Maček in sod., 2019, Davison in sod., 2021). Trenutno poznamo več kot 300 morfotipov AM gliv, določenih na podlagi morfologije spor (http:// www.amf-phylogeny.com/). Na podlagi molekulskih analiz lahko ocenimo, da je število različnih taksonov AM gliv v okolju bistveno večje, kot kažejo taksonomske študije na podlagi morfoloških znakov. Virtualna taksonomija (virtualni taksoni – VT) AM gliv, ki je bila vzpostavljena v specializirani bazi podatkov s področja raziskav AM gliv, ki se imenuje MaarjAM (Öpik in sod., 2010), tudi bazira na uporabi genskega markerja 18S rRNA. Enote, definirane znotraj tega sistema, so t.i. virtualni taksoni (VT), ki po navedbah avtorjev baze MaarjAM verjetno predstavljajo monofiletske skupine AM gliv, pri katerih podobnost sekvenc znotraj skupine presega dogovorno določeno mejo 97 %. Resolucija VT naj bi približno ustrezala tisti, ki definira vrste AM gliv, določene na podlagi morfoloških znakov (Öpik & Davison, 2016). Baza MaarjAM (http://maarjam.botany.ut.ee) vsebuje več kot 450 virtualnih taksonov, identificiranih na podlagi sekvenc za malo podenoto ribosoma (18S rRNA) (Öpik in sod., 2014), vendar se številke spreminjajo oz. so večje, odvisno, katere genske označevalce uporabimo za identifikacijo taksonov. Klasifikacija sekvenc genov 18S rRNA z uporabo virtualnih taksonov (VT), ki so določeni na podlagi baze MaarjAM (Öpik in sod., 2009, 2010), nam omogoča poenoten sistem poimenovanja genotipov, ki jih lahko v ekoloških študijah uporabimo za najboljši približek identifikaciji do nivoja vrst oz. rodov (Öpik in sod., 2014). Pogosta kritika uporabe molekulskih metod za številčno ovrednotenje mikrobnih združb so tudi napake posamezne metode, ki lahko vplivajo na končni rezultat analize. Ena izmed najpogosteje omenjenih napak je povezana z metodo PCR in neenakomernim pomnoževanjem DNA različnih taksonov znotraj združbe (t.i. PCR bias ali PCR pristranskost). PCR je ključni del praktično vseh molekulskih pristopov za analizo mikrobnih 6 Acta agriculturae Slovenica, 118/3 – 2022 združb, vključno z združbami AM gliv. Znotraj izolata DNA iz vzorca korenin namreč glivna DNA predstavlja le majhen del celokupne ekstrahirane DNA, zato je potrebno specifično pomnoževanje (amplifikacija) regij DNA, ki so informativne za ločevanje različnih taksonov AM gliv. Odvisno od ciljev in namena raziskave se za to lahko uporablja različne genske označevalce, najpogosteje pa je v ekoloških raziskavah AM gliv uporabljena že omenjena regija 18S rRNA. Potencialne napake oz. pristranskost metode PCR za pomnoževanje specifičnih taksonov (genotipov) AM gliv ter primernost uporabe presejalne metode polimorfizma dolžine terminalnih restrikcijskih fragmentov (TRFLP) za kvantitativne raziskave je bila za regijo 18S rRNA AM gliv testirana v študiji Cotton in sod. (2014). V raziskavi so potrdili, da pri uporabi metod PCR za gene 18S rRNA ne prihaja do bistvenih razlik v pomnoževanju DNA med različnimi genotipi AM gliv in se zato te metode lahko uporablja tudi za kvantitativne analize združb AM gliv, zavedati pa se moramo določenih omejitev. Napake v pomnoževanju regij DNA z metodo PCR se lahko zgodijo, če se različni genotipi pomnožujejo različno hitro zaradi razlik v njihovih nukleotidnih zaporedjih ali zaradi same kinetike reakcije pomnoževanja (Kanagawa, 2003). Argument, da se to ne pojavlja pri AM glivah ob uporabi najpogosteje uporabljenih začetnih oligonukleotidov AM1 in NS31 je ta, da so regije DNA, na katere se ti oligonukleotidi vežejo zelo konzervativne (ohranjene med sorodstveno oddaljenimi taksoni) in imajo obenem zelo majhno variabilnost v vsebnosti baz gvanina (G) in citozina (C) v amplikonih (Dumbrell in sod., 2010). Odsotnost tovrstnih napak (pristranskosti pri pomnoževanju med različnimi genotipi) pri reakciji PCR, je eden izmed osnovnih pogojev, da je neka analiza lahko kvantitativna in obenem omogoča oceno različnih indeksov pestrosti oz. diverzitetnih indeksov (Cotton in sod., 2014). V nadaljnjih testih so uporabili tudi analizo TRFLP za karakterizacijo umetno ustvarjene združbe z znanimi razmerji količin vhodne DNA različnih genotipov AM gliv. Podatki študije kažejo, da je protokol TRFLP v kombinaciji s PCR močno in konsistentno orodje za analizo združb AM gliv, medtem, ko na nivoju analiz populacij posameznih taksonov (genotipov) avtorji študije priporočajo več previdnosti (Cotton in sod., 2014). Ta eksperiment je torej potrdil hipotezo, da je možno z uporabo PCR in metodo TRFLP ustrezno ugotoviti relativne razlike med različnimi združbami AM gliv, v smislu njihove diverzitete in sestave. Študija je tudi pokazala, da pri pomnoževanju različnih genotipov AM gliv za gene 18S rRNA metoda PCR posameznih genotipov ne pomnožuje bolj, kot drugih, kar pomeni, da se ta metoda lahko uporablja tudi v sklopu drugih kvantitativnih analiz združb AM gliv, kjer predstavlja metoda PCR pomemb- Razvoj raziskovalnih metod za karakterizacijo združb arbuskularnih mikoriznih gliv in potencialni vpliv ... na vegetacijo no komponento. Mednje vsekakor sodijo tudi vse metode sekvenciranja naslednjih generacij (NGS), ki so danes za raziskave ekologije združb AM gliv največ v uporabi in katerih sestavni del je tudi pomnoževanje sekvenc s PCR (npr. platforma Illumina). 2.1 UPORABA KLONIRANJA IN DOLOČANJA NUKLEOTIDNEGA ZAPOREDJA PO SANGERJU TER PRESEJALNIH METOD V času nastanka prvega preglednega članka o uporabi molekulskih pristopov pri raziskavi AM gliv (Maček, 2009) se je za raziskave sestave mikrobnih združb, vključno z združbami rizosfernih AM gliv iz okoljskih vzorcev (tal ali korenin), v veliki meri uporabljalo molekulske pristope, ki so temeljili na postopkih kloniranja in določanja nukleotidnega zaporedja po Sangerju. Če povzamemo na kratko, tak pristop običajno vsebuje naslednje korake: (1) pomnoževanju glivne DNA z reakcijo PCR, (2) ‚in vivo‘ ločevanje pomnoženih fragmentov DNA s kloniranjem pomnožkov (amplikonov) v plazmide in uporabo kolonij bakterij vrste Escherichia coli za ločevanje sekvenc, (3) ponovna/sekundarna reakcija PCR za namen ločevanja sekvenc, potrebnega za uporabo (4) sekventorjev prve generacije (sekvenciranje po Sangerju) (glej opis postopka v Maček, 2009). Vsi našteti koraki, predvsem pa kloniranje in uporaba celičnih kultur, so predstavljali velik časovni in finančni zalogaj, zato je bilo število vzorcev (biološke ponovitve), kot tudi število sekvenc, ki so bile sekvencirane za posamezni vzorec (globina sekvenciranja), zelo omejeno. Za posamezno okoljsko študijo sestave združbe AM gliv smo na tak način tipično lahko pregledali nekje med 500 in 1000 sekvenc za posamezni genski marker (npr. fragment 18S rRNA). Posledica tega je bila, da so bile združbe opisane zelo pomanjkljivo. Lahko se namreč zgodi, da na ta način nevede izpustimo tiste taksone, ki so znotraj združbe manj pogosti (redki), združbo pa zaradi metodoloških omejitev opisujemo zgolj na podlagi bolj dominantnih in bolj številčnih predstavnikov skupine. Vmesna faza med zgoraj opisanim postopkom in novimi postopki sekvenciranja naslednjih generacij (NGS) je bila uporaba različnih presejalnih metod, ki temeljijo na primerjavah t.i. prstnih odtisov združbe oz. ločevanju produktov PCR z dvojno vijačnico podobne dolžine, vendar z različno sekvenco. Te metode so omogočile zajem nekoliko večjega števila vzorcev in tudi večjo globino vzorčenja (število obravnavanih sekvenc). Mednje sodijo metode, kot so denaturacijska gradientna gelska elektroforeza (DGGE), temperaturna gradientna gelska elektroforeza (TGGE) in že omenjeni polimorfizem dolžine terminalnih restrikcijskih fragmentov (TR- FLP). Med naštetimi metodami ima TRFLP največjo zmogljivost v smislu možnosti obdelave večjega števila bioloških vzorcev, medtem, ko so tehnike gelske elektroforeze (DGGE, TGGE) zamudne in omogočajo obdelavo le manjšega števila vzorcev. To pomeni, da so manj uporabne za ekološke študije, kjer je med vzorci običajno velika variabilnost in je zato potrebno v študijo vključiti veliko število vzorcev. Slaba stran vseh teh metod je tudi ta, da so primarno presejalne narave, zato v končni fazi običajno nimamo vpogleda v sekvence posameznega markerskega gena raziskovanih organizmov. Tako metodo lahko zato uporabljamo samo za relativne primerjave sestave združb in biodiverzitete med primerjanimi vzorci znotraj ene študije, ne omogočajo pa primerjave različnih študij med sabo. Tako je bila pred razvojem in širšo uporabo metod NGS relativno pogosto uporabljana analiza za relativne primerjave sestave združb organizmov iz okoljskih vzorcev preučevanje polimorfizma dolžine terminalnih restrikcijskih fragmentov (TRFLP) tarčnih genov (prokariontskih 16S ali evkariontskih 18S rRNA), ki v nekaterih primerih omogoča tudi kvantitativne študije. Metoda je bila pogosto uporabljana tudi na področju mikrobne ekologije in okoljske mikrobiologije, predvsem zaradi cenovne dostopnosti, filogenetske ločljivosti in enostavne analize večjega števila vzorcev. Analiza TRFLP ima torej dovolj veliko zmogljivost (omogoča obdelavo zadostnega števila vzorcev), da lahko z njo preučimo tudi vplive različnih okoljskih dejavnikov na strukturo in dinamiko mikrobnih združb. Na tak način dobimo t.i. ‚prstni odtis‘ (finger-print) združbe raziskovane skupine organizmov za analizirane vzorce, zato tehniko TRFLP imenujemo tudi tehnika prstnih odtisov. Take podatke lahko uporabimo za analizo diverzitete (izračun različnih indeksov raznolikosti) in sestave združbe, pri čemer lahko izvajamo primerjave med vzorci, zajetimi znotraj ene študije, ne pa tudi med različnimi študijami, kar je pomanjkljivost tehnik, ki uporabljajo t. i. tehniko prstnih odtisov. Poznati moramo tudi specifike posamezne preučevane skupine organizmov in omejitve uporabe posamezne metode za to skupino. Primernost metode TRFLP so testirali tudi za kvantitativne analize združb AM gliv, pri tem pa avtorji študije opozarjajo, da tehnika TRFLP v vseh testiranih vzorcih precenjuje vrstno pestrost AM gliv znotraj raziskovane združbe, zato je pri kvantitativnem vrednotenju indeksov raznolikosti potrebna pozornost pri interpretaciji s to tehniko pridobljenih rezultatov (Cotton in sod., 2014). Sama metoda TRFLP ne producira celotnih sekvenc posameznih organizmov znotraj združbe, se pa lahko s TRFLP pridobljeni podatki primerjajo z obstoječo bazo sekvenc, če želimo pridobiti podatke o identiteti posmeznega vzorca (Dickie & FitzJohn, 2007, Roberts in sod., 2012). Danes so tehniko TRFLP v raziskavah ekologije združb mikActa agriculturae Slovenica, 118/3 – 2022 7 I. MAČEK roorganizmov v veliki meri nadomestile tehnike NGS, katerih velika prednost je, da končne izhodne podatke predstavljajo celotne sekvence posameznih amplikonov, kar omogoča lažjo in neposredno identifikacijo organizmov, ki pa je odvisna od informativnosti regije, ki jo sekvenciramo za posamezno skupino organizmov. Kot že rečeno, naj bi regija 18S rRNA pri AM glivah približno ustrezala nivoju morfološke vrste, se pa to področje še zelo intenzivno raziskuje in lahko v prihodnosti pričakujemo novosti. 2.2 DOLOČANJE NUKLEOTIDNEGA ZAPOREDJA (SEKVENCIRANJE NASLEDNJIH GENERACIJ – NGS) Metode NGS so najprej uporabljali v medicini in pri poskusih sekvenciranja človeka in drugih primatov (Wheeler in sod., 2008). Relativno hitro so se te metode razširile tudi na področje ekoloških raziskav in raziskav mikrobnih združb (mikrobiomov) v različnih okoljih, saj so omogočale vključevanje bistveno večjega števila vzorcev v raziskavo in obenem bistveno večjo globino sekvenciranja znotraj posameznega vzorca (najprej od več sto tisoč pa vse do več sto milijonov sekvenc pri danes uporabljanih pristopih). Ti novi principi so močno vplivali na številna področja v mikrobni ekologiji, okoljski mikrobiologiji, ekologiji tal in raziskavah rizosfere, kot tudi pri raziskavah različnih interakcij med organizmi, vključno z interakcijami rastlin z drugimi organizmi (npr. mikoriza). Danes je tudi v okoljskih raziskavah pogosta praksa pomnoževanja tarčnih filogenetskih in/ali funkcionalnih genskih označevalcev in obenem uporaba pristopov NGS za karakterizacijo njihove raznolikosti. Nova tehnologija omogoča vse številčnejše sete vzorcev, kar je bistvenega pomena za ekološke raziskave, ki se lahko izvajajo v različnih dimenzijah, tako prostora, kot tudi časa (vzorčenja v več prostorskih in časovnih točkah, raziskave dinamike procesov in sukcesije). Zavedati pa se moramo, da so prav vsi pristopi NGS podvrženi metodološkim napakam oz. so lahko pristranski, pri čemer gre na eni strani za produkcijo visoko kvalitetnih podatkov, ki jih zahtevajo raziskave, in na drugi napačnih sekvenc ter metodološkega šuma. Vsi pristopi z uporabo NGS zato zahtevajo natančno bioinformacijsko analizo in procesiranje podatkov, kar omogoča kvalitativno filtriranje in procesiranje sekvenc z namenom izogibanja zavajajočih interferenc iz metodoloških napak. Podobna previdnost je potrebna tudi pri vseh nadaljnjih statističnih analizah pridobljenih podatkov, saj lahko nepravilen izbor statističnega pristopa rezultira v napačnih zaključkih raziskave. 8 Acta agriculturae Slovenica, 118/3 – 2022 Prav s tem namenom je nastalo tudi kar nekaj preglednih objav oz. pregleda metodoloških pristopov, ki sistematično podajajo navodila za lažje spopadanje z izzivom obdelave podatkov, ki izhajajo iz NGS (npr. za osnovno analizo podatkov, ki izhajajo iz sekvenciranja amplikonov z namenom raziskav raznolikosti in ekologije združb AM gliv (Dumbrell in sod., 2017). Tipično bioinformacijski pristopi vključujejo metode za preverjanje kakovosti sekvenc in odstranjevanja šuma, formiranje operacijskih taksonomskih enot (Operational Taxonomic Unit – OTU), taksonomsko določanje posameznih sklopov sekvenc ter osnovne statistične analize za testiranje hipotez. Prikaz teh metod za dva pogosto uporabljena pristopa (QIIME in mothur), skupaj s samostojnimi orodji (vključno z odprtokodnim programskim okoljem ter jezikom R), so predstavljene v objavi Dumbrell in sod. (2017), kjer so predstavljeni pristopi za obdelavo podatkov sekvenciranja amplikonov, ki izhajajo iz dveh pogosto uporabljenih tehnologij NGS, v preteklosti več uporabljane tehnologije 454-pirosekvenciranja ter danes široko uporabljane platforme Illumina. Zaradi hitrega napredka tehnologije in z njo povezane bioinformatike pa je nujno redno spremljanje tekočih objav in drugih virov na to temo. Zaradi obsežnosti in kompleksnosti podatkov, ki izhajajo iz tehnik sekvenciranja amplikonov z NGS je pogost izziv tudi njihova ustrezna predstavitev. Izziv, ki se skriva v novih tehnologijah je predvsem ta, da nas lahko zaslepi analitična moč novih metod, obenem pa je naše zavedanje o tem, da zaradi hitrega razvoja teh pristopov lahko pridemo tudi do napačnih zaključkov, pomanjkljivo. Napačni zaključki so lahko rezultat napak v sami tehnologiji, pomanjkljivega testiranja metod in/ ali pomanjkanja izkušenj. Nujno je, da nek problem oz. študijo že v začetni fazi načrtovanja naslovimo z jasnimi vprašanji, posledica katerih so tudi jasno zastavljene raziskovalne hipoteze in ciljni metodološki pristopi, ki izhajajo iz teh hipotez. Vsekakor pa predstavljajo metode NGS ob pravilni uporabi močno analitično orodje, ki odpira povsem nove možnosti v raziskavah ekologije tako bolj vidnih in karizmatičnih nadzemnih organizmov, kot tudi bolj skritih, a pomembnih akterjev v podzemnem delu kopenskih ekosistemov. 3 VPLIV OKOLJSKIH IN GEOGRAFSKIH DEJAVNIKOV NA SESTAVO ZDRUŽB AM GLIV IN NJIHOVO RAZŠIRJENOST Znano je, da sestavo združb AM gliv določajo različni dejavniki okolja, vključno s klimatskimi in talnimi specifikami, ki delujejo tako na lokalni, kot tudi na globalni ravni (npr. Dumbrell in sod., 2010, Kivlin in sod., Razvoj raziskovalnih metod za karakterizacijo združb arbuskularnih mikoriznih gliv in potencialni vpliv ... na vegetacijo 2011, Lekberg in sod., 2011, Maček in sod., 2011, Hazard in sod., 2013, Davison in sod., 2015, Maček in sod., 2019, Vetrovsky in sod., 2019, Davison in sod., 2021). Na splošno je pri pojavljanju različnih taksonov AM gliv še vedno relativno malo dostopnih informacij na ravni odnosa posameznega organizma in okolja (Davison in sod., 2020). Po podatkih iz številnih študij, ki so zbrane v bazi MaarjAM (Öpik in sod., 2010) lahko vidimo, da so mnogi virtualni taksoni (VT) AM gliv široko geografsko razširjeni in se pojavljajo v številnih habitatnih tipih (Davison in sod., 2015, Savary in sod., 2018). Taka opažanja pa so nastala večinoma na podatkih o prisotnosti taksonov v določenem habitatu, ni pa podatkov v kolikšni meri številčnost (abundanca) posameznih VT variira vzdolž gradienta določenega abiotskega dejavnika. Taksone AM gliv so v preteklosti klasificirali v različne ekotipe (Alzarhani in sod., 2019) ter v ekološke skupine, kot so generalisti in specialisti na osnovi različnih rangov, ki vključujejo geografske dejavnike (Moora in sod., 2011, Bouffaud in sod., 2016), habitat (Sykorova in sod., 2007, Oehl in sod., 2010, Vályi in sod., 2015) ali rastlinske gostiteljske vrste (Helgason in sod., 2007). Poudariti pa je treba, da je tovrstno klasificiranje taksonov v posamezne ekološke niše omejeno na obseg okoljskih dejavnikov, ki jih pokriva posamezna študija in posplošenje tega pojava ni mogoče. V nekaterih novejših objavah poročajo, da so si določene funkcionalne lastnosti med sorodnimi morfološkimi vrstami AM gliv podobne (Powell in sod., 2009, Hoeksema in sod., 2018). Dejstvo pa je, da predstavljajo morfološko opisane vrste AM gliv, identificirane na podlagi morfologije celične stene njihovih spor, le majhen del raznolikosti AM gliv, ki so bile določene na osnovi molekulskih označevalcev (markerskih genov) (Öpik in sod., 2014). V zelo obsežni nedavni študiji, ki je vključevala več kot 300 talnih vzorcev iz različnih naravnih ekosistemov iz celega sveta so ugotovili, da so porazdelitev VT v različnih ekosistemih skupaj pojasnile tako okoljske, kot tudi prostorske (geografske) variable. Med njimi sta bili temperatura okolja in vrednost pH tal najpomembnejša okoljska določevalnika porazdelitve in lokalne relativne abundance (številčnosti) različnih taksonov AM gliv (Davison in sod., 2021). V študiji so ugotovili tudi različne vzorce ekoloških niš, ki so bili najbolj opazni na ravni družin AM gliv, kar kaže, da sorodni taksoni oz. VT zavzemajo podobne ekološke niše. Za predstavnike družine AM gliv Acaulosporaceae je tako značilen optimum pri manjši vrednosti pH tal, in nižji temperaturi, medtem, ko so predstavniki družine Gigaporaceae bolj številčni v bolj vlažnih območjih z več dežja (Davison in sod., 2021). Na to temo pa bo v prihodnjem desetletju sigurno še veliko novih podatkov, tudi v luči vpliva klimatskih sprememb na AM glive in njihovo simbiozo z rastlinami (Maček in sod., 2019). 4 ZAKLJUČEK Razumevanje vpliva globalnih sprememb na terestrične ekosisteme zahteva povezovalen pristop med različnimi disciplinami, ki raziskuje odzive skozi vse ravni biološke organizacije in skozi različne prostorsko-časovne skale. Obenem mora ta pristop vključevati tako nadzemno, kot tudi podzemno raznolikost znotraj ekosistemov, saj sta obe neločljivo povezani in prepleteni. V večini primerov še vedno ni celostnega razumevanja odziva interakcij komponent nadzemne in podzemne raznolikosti na akutne kratkoročne (npr. suša, toplotni valovi) ter kronične dolgoročne globalne in klimatske spremembe (npr. segrevanje, povečana koncentracija CO2, onesnaženje). Prav zato so nujno potrebni eksperimenti, ki naslavljajo te vrzeli v razumevanju delovanja ekosistemov. Nove metode sekvenciranja omogočajo veliko ponovljivost in s tem dokaj robusten pristop k raziskavam v ekologiji, predvsem v smislu velikega števila bioloških ponovitev (zadosti ponovitev za ustrezno analizo podatkov), obravnavo vseh ravni ekoloških združb (nadzemnega in podzemnega – rizosfernega), in v smislu primernega trajanja eksperimenta (zaželene so dolgoročne študije, ki zajemajo vzorčenje preko več sezon ali celo let). Slednje je nujno za razumevanje in ločevanje dolgoročnih in kratkoročnih odzivov na vseh ravneh biodiverzitete. Najnovejša molekulska orodja za raziskave v ekologiji združb, kot so metode NGS, ki omogočajo pridobivanje podatkov o sekvencah organizmov iz različnih okolij, postavljajo vse cenejše in široko dostopne. Pri tem pa je nujno zavedanje, da kljub široki dostopnosti, kvalitetna biološka interpretacija molekulske karakterizacije združb posameznih skupin organizmov zahteva veliko specialističnega znanja o specifični skupini organizmov. Zavedati se moramo na primer, da tako tehnika TRFLP, kot tudi NGS, predstavljata analize relativne številčnosti (abundance) taksonov znotraj vzorcev, zato ju ne smemo uporabljati za ugotavljanje razlik v absolutni številčnosti med posameznimi vzorci. Na koncu se moramo zavedati tudi dejstva, da lahko podajajo ocene sestave združb, ki temeljijo na ekstraktih DNA, le podatke o genski raznolikosti in genski sestavi združb. Nemogoča je namreč ocena biomase posameznih vrst teh organizmov v nekem okolju na osnovi takih podatkov zaradi medvrstne in časovne variabilnosti števila kopij posameznih genov na enoto rasti, kar posebej velja za nitaste organizme, kot so AM glive (Corradi in sod., 2007, Jansa in sod., 2008). Bistveno je torej, da se obenem zavedamo tako moči tehnologije, da v kratkem času producira ogromno količino podatkov, kot tudi vseh omejitev in novosti, vključno s hitro spreminjajočimi se postopki bioinformatike, ki je neločljivo povezana z vsakim postopkom NGS. Tak prisActa agriculturae Slovenica, 118/3 – 2022 9 I. 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CERN-TH-2022-137 DESY-22-135 1 INTRODUCTION Azimuthal di-jet correlations with parton branching TMD distributions A. Bermudez Martineza and F. Hautmann b,c,d a Deutsches Elektronen-Synchrotron DESY, Notkestr. 85, 22607 Hamburg, Germany b CERN, Theoretical Physics Department, CH 1211 Geneva 23, Switzerland c Elementaire Deeltjes Fysica, Universiteit Antwerpen, B 2020 Antwerpen, Belgium d RAL, Chilton OX11 0QX and University of Oxford, Oxford OX1 3PU, UK Presented at DIS2022: XXIX International Workshop on Deep-Inelastic Scattering and Related Subjects, Santiago de Compostela, Spain, May 2-6 2022 Abstract The parton branching formulation of TMD evolution has recently been used to make predictions for jet observables at the Large Hadron Collider (LHC), including perturbative matching at next-to-leading order (NLO). This contribution presents results for the azimuthal ∆φ correlations in events with di-jets at large transverse momentum. It focuses on the back-to-back region of large ∆φ and discusses prospects for detailed studies of QCD dynamics in this region at the LHC. 1 Introduction Azimuthal correlations between two jets have been measured at the LHC by the ATLAS and CMS collaborations [1, 2, 3, 4, 5]. A detailed understanding of these correlations is important for studies of the Quantum Chromodynamics (QCD) sector of the Standard Model (SM) and searches for Beyond-the-SM (BSM) scenarios with di-jet signatures. At leading order (LO) in the strong coupling αs , two jets are produced back-to-back, i.e., with azimuthal angle ∆φ = π. Deviations from this configuration measure higher-order QCD radiation. In the region near ∆φ = π this is primarily soft gluon radiation, while in the region of small ∆φ it is primarily hard QCD radiation. Since initial-state parton radiation moves the jets away from the ∆φ = π region, it is relevant to investigate the influence of transverse momentum recoils in the QCD showers [6, 7, 8], taken into account via transverse momentum dependent (TMD) [9] parton distributions, on the description of the ∆φ measurements. In this article we discuss this by using the Parton Branching (PB) approach [10, 11] to TMD distributions. This approach has successfully been used at next-to-leading order (NLO) to extract TMD parton distributions [12] from precision deep-inelastic data [13] using xFitter [14, 15] (results are available from the repository [16, 17]). It has also been successfully used to make predictions for Drell-Yan transverse momentum spectra both at the LHC [18] and in lower energy experiments [19]. We here apply this approach to di-jet production, presenting results from the work in Ref. [20]. We compute predictions for di-jet azimuthal correlations, using the PB TMD evolution matched with NLO perturbative matrix elements. In the region near the back-to-back configuration the QCD Sudakov process depends on the soft function [21]. Unlike the case of Drell-Yan di-lepton production, factorization breaking effects [22, 23, 24] can arise in the case of di-jets due to long-timescales soft-gluon correlations between initial and final states. We examine the possibility of investigating these effects with high transverse momentum jets at the LHC. 1 2 NLO MATCHING WITH PB TMD dσ/d∆φ( jj) [pb/rad] Dijet 10 8 MCatNLO+CAS3 MCatNLO(fNLO) MCatNLO(LHE) 10 7 10 6 10 5 10 4 10 3 10 2 0 0.5 1 1.5 2 2.5 3 ∆φ( jj) Figure 1: Azimuthal ∆φ distributions obtained from the fixed NLO calculation (MCatNLO(fNLO)), the (unphysical) LHE level (MCatNLO(LHE)), and after inclusion of PB TMDs (MCatNLO+CAS3) [20]. The article is organized as follows. In Sec. 2 we briefly describe the main elements of the PB TMD calculation at NLO. In Sec. 3 we illustrate the results for di-jet azimuthal distributions, and compare them with LHC experimental measurements. Conclusions are given in Sec.4. 2 NLO matching with PB TMD The PB approach [10, 11] provides evolution equations for TMD distributions in terms of Sudakov form factors and splitting probabilities, and a corresponding TMD parton shower in a backward evolution scheme. PB TMD distributions and parton showers are implemented in the Monte Carlo event generator CASCADE3 [25]. A method to match TMD evolution with NLO perturbative matrix elements has been developed for the case of the Drell-Yan process in Refs. [18, 19] using the framework of MAD GRAPH5_AMC@NLO [26]. We next apply this method to the case of the jet production process [20], matching PB TMD distributions and parton showers with di-jet NLO matrix elements from MADGRAPH5_AMC@NLO. Further details on the NLO matching method with PB TMD are given in Ref. [27], where a comparison of MCatNLO+CASCADE3 [25] and MCatNLO+HERWIG6 [28] matching is performed. Fig. 1 illustrates the result of applying the matching method. It shows the differential distribution in the azimuthal angle ∆φ between the two leading jets as obtained from the calculations at fixed NLO (blue curve), at the (unphysical) level including the subtraction terms from the matching (LHE level, green curve), and after inclusion of PB TMDs (red curve). We observe the rising cross section of the fixed NLO calculation towards large ∆φ (corresponding to the divergent behavior of the NLO calculation in the back-to-back configuration), the decay towards large ∆φ once the subtraction terms are included, and the smooth prediction once the TMD distributions and showers are included. 2 3 [deg−1] Data MCatNLO+CAS3(Scale7pt) MCatNLO+P8(Scale7pt) noMPI MCatNLO+P8(Scale7pt) MPI b 1 leading CMS, 13 TeV, ∆φ12 inclusive 2-jet, 200 < pT < 300 GeV b b b b 1 dσ σ d∆φ12 1 dσ σ d∆φ12 [rad−1] leading CMS, 13 TeV, pp → two jets, 200 < p T DI-JET AZIMUTHAL DISTRIBUTIONS b b b b 0.09 Data MCatNLO+CAS3(Scale7pt) MCatNLO+P8(Scale7pt) noMPI MCatNLO+P8(Scale7pt) MPI b 0.08 0.07 b b b b 10−1 < 300 GeV b b b 0.06 b b b b 0.05 b 10−2 b b b b b b 0.04 b b 1.6 b b 1.8 b b b 2 b b 2.2 b b 2.4 b b 2.6 b b b 2.8 b b MC/Data MC/Data b 1.4 1.3 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 b 3 ∆φ12 [rad] 1.15 1.1 1.05 1 0.95 0.9 0.85 0.8 170 b b b 172 b b 174 b b 176 b b 178 b 180 ∆φ12 [deg] Figure 2: Azimuthal correlation over a wide ∆φ range (left) and in the backto-back region (right) [20]. CMS data [4, 5] are compared with results from MCatNLO+PYTHIA8 and MCatNLO+CAS3. 3 Di-jet azimuthal distributions We now use the method of the previous section to compute NLO-matched PB TMD predictions for di-jet distributions in the phase space of the CMS measurements [4, 5]. We consider selection cuts for leading jets with transverse momentum p T > 200 GeV and p T > 1000 GeV. With this event selection one is able to explore TMD dynamical effects over a broad range both in the transverse momentum of the TMD distribution, set by the p T imbalance between the jets, and in its evolution scale, set by the hard scale of the event, e.g. the leading jet p T . In particular, in a neighborhood of order 0.1 rad from ∆φ = π, the p T imbalance ranges from a few ten GeV for the highest p T jets down to few GeV. At large p T imbalance, the evolution of the transverse momentum is dominated by perturbative contributions to the evolution kernels and can be explored through directly measurable jets, while at lower p T imbalance both perturbative and non-perturbative components can be investigated. In Fig. 2 we report the NLO-matched PB TMD results (labelled MCatNLO+CAS3), together with CMS data [4, 5] and collinear shower calculations from MCatNLO+PYTHIA8 [29]. The shape of the ∆φ distribution is different between the TMD and collinear shower calculations, emphasizing the relevance of the detailed dynamics of QCD shower evolution. The uncertainty bands on the MCatNLO+CAS3 predictions are obtained from scale variations and TMD uncertainties [20]. The uncertainty bands on the MCatNLO+PYTHIA8 predictions are obtained from scale and associated shower variations according to the method of [30] together with the guidelines of [31]. For the MCatNLO+PYTHIA8 calculation the effect of multi-parton interactions (MPI) is also shown, using the parameters of the tune CUETP8M1 [32]. For leading jet p T > 200 GeV, the MPI effect is not large. MCatNLO+CAS3 describes the measurements well at large and intermediate ∆φ. In the decorrelated region at low ∆φ a deficit is observed. This is due to missing higher-order contributions from multiple QCD emissions beyond NLO. To take these contributions into account, one needs to go beyond the framework of the present calculation, for example by employing TMD multi-jet merging techniques [33, 34]. In Fig. 3 we focus on the large ∆φ region of nearly back-to-back jets. This region is of special interest, as possible factorization-breaking effects have long been conjectured to arise for back-to-back jets due to soft-gluon interactions between initial and final states. We see from Fig. 3 that the measurements are well described by the MCatNLO+CAS3 predictions. Only in 3 4 leading [deg−1] 0.09 Data MCatNLO+CAS3(Scale7pt) MCatNLO+CAS3(TMD unc) b 0.08 leading CMS, 13 TeV, ∆φ12 inclusive 2-jet, 1000 < pT < 300 GeV 1 dσ σ d∆φ12 1 dσ σ d∆φ12 [deg−1] CMS, 13 TeV, ∆φ12 inclusive 2-jet, 200 < pT b b 0.07 CONCLUSION Data MCatNLO+CAS3(Scale7pt) MCatNLO+CAS3(TMD unc) b 0.2 < 1200 GeV b 0.15 b b b 0.06 0.1 b 0.05 b b b b b 0.05 b 0.04 b b b b b b 172 b b 174 b b 176 b b 178 MC/Data MC/Data 1.15 1.1 1.05 1 0.95 0.9 0.85 0.8 170 b b b b b 180 ∆φ12 [deg] 1.15 1.1 1.05 1 0.95 0.9 0.85 0.8 170 b b b 172 b b 174 b b 176 b b 178 b 180 ∆φ12 [deg] Figure 3: Azimuthal correlation in the back-to-back region for leading jet p T > 200 GeV (left) and p T > 1000 GeV (right) as measured by CMS [5] compared with predictions from MCatNLO+CAS3 [20]. Shown are the uncertainties coming from the scale variation as well as the uncertainties coming from the TMD. the highest bin (∆φ > 179o ) a deviation of about 10% is observed. Detailed phenomenological studies in this region are warranted, using fine binning in angle. As discussed in Ref. [27], further insight may be gained from the combined analysis of ∆φ correlations in di-jet and Z-boson + jet events. At low p T the boson-jet state is more strongly correlated azimuthally than the jet-jet state, while for p T far above the electroweak scale the behaviors become more similar. This can be connected to features of the partonic initial-state and final-state radiation in the boson-jet and jet-jet cases. Initial-state and final-state radiation (see the recent studies [35, 36] in the Z + jet process) may give rise to color interferences and potential factorization-breaking effects [22, 24, 37]. If so, different breaking patterns can be expected for strong and weak azimuthal correlations, influencing differently the boson-jet and jet-jet cases. Systematic measurements of di-jet and Z-boson + jet distributions are thus proposed [27], scanning the phase space from low transverse momenta p T ≈ O(100 GeV) to high transverse momenta p T ≈ O(1000 GeV). Fig. 4 illustrates another aspect of the QCD dynamics in the back-to-back region, namely, the sensitivity to soft-gluon angular ordering [38, 39, 40, 41] in the TMD evolution [11, 42]. The MCatNLO+CAS3 curves labelled Set1 and Set 2 in Fig. 4 refer to two sets of PB TMD distributions [12] differing by the scale in the QCD running coupling: Set 2 fulfills the soft-gluon angular ordering conditions by using the transverse momentum emitted at each branching as a scale for αs , while Set 1 uses the branching scale as a scale for αs , as in DGLAP ordered evolution. We see that the shape of the azimuthal correlation is sensitive to angular ordering effects in the back-to-back region. Set 2 provides a better description of the measurements in this region. 4 Conclusion In this article we have discussed predictions from PB TMD parton showers for final state observables in jet production at the LHC, focusing on the azimuthal correlations of jets with large transverse momenta. The PB TMD shower matched with NLO calculations provides a good description of experimental measurements of di-jet production at the LHC in the correlation region of high 4 REFERENCES REFERENCES leading 1 dσ σ d∆φ12 [deg−1] CMS, 13 TeV, ∆φ12 inclusive 2-jet, 200 < pT 0.1 < 300 GeV Data MCatNLO+CAS3(Set1) MCatNLO+CAS3(Set2) b 0.09 0.08 b b 0.07 b b 0.06 b 0.05 b b b 0.04 b MC/Data b 1.15 1.1 1.05 1 0.95 0.9 0.85 0.8 170 b b b 172 b b 174 b b 176 b b 178 b 180 ∆φ12 [deg] Figure 4: Impact of the transverse momentum q T scale in the running coupling at large ∆φ [20]. 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Journal of Clinical Medicine Article Prevalence of Function-Dependent Temporomandibular Joint and Masticatory Muscle Pain, and Predictors of Temporomandibular Disorders among Patients with Lyme Disease Magdalena Osiewicz 1, * , Daniele Manfredini 2 , Grażyna Biesiada 3 , Jacek Czepiel 3 , Aleksander Garlicki 3 , Ghizlane Aarab 4 , Jolanta Pytko-Polończyk 1 and Frank Lobbezoo 4 1 2 3 4 * Department of Integrated Dentistry, Dental Institute, Faculty of Medicine, Jagiellonian University Medical College, 31-155 Krakow, Poland School of Dentistry, University of Siena, 53100 Siena, Italy Department of Infectious and Tropical Diseases, Faculty of Medicine, Jagiellonian University Medical College, 31-155 Krakow, Poland Department of Orofacial Pain and Dysfunction, Academic Centre forDentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, 1081 LA Amsterdam, The Netherlands Correspondence: Magdalena.osiewicz@uj.edu.pl Received: 28 May 2019; Accepted: 27 June 2019; Published: 28 June 2019



 Abstract: The aim was to determine the occurrence of temporomandibular disorders (TMDs) in patients with Lyme disease (LD), and to estimate the contribution of factors that may identify TMD among LD patients. In seventy-six (N = 76) adult patients with LD (mean age 57.6 ± 14.6 years) and 54 healthy non-Lyme volunteers with a mean age of 56.4 ± 13.5 years, possible function (i.e., non-pain) diagnoses were established using the Research Diagnostic Criteria of Temporomandibular Disorders (RDC/TMD). Pain diagnoses were established by means of the function-dependent dynamic and static tests. The two groups did not significantly differ in the frequency of disc displacements diagnoses and function-dependent pain diagnoses. LD showed a significantly higher frequency (p < 0.001) of osteoarthrosis than the control group. For the prediction of pain diagnoses in LD patients, the single regression analyses pointed out an association with age, sleep bruxism (SB), and awake bruxism (AB). Two predictors (i.e., SB (p = 0.002) and AB (p = 0.017)) were statistically significant in the final multiple variable model. The frequency of TMD in patients with LD based on function-dependent tests was not significantly different from that in the control group. This investigation suggests that the contribution of bruxism to the differentiation between patients with Lyme and TMD is high. Keywords: palpation tests; temporomandibular disorders; static/dynamic tests; comorbidity; Lyme disease 1. Introduction Lyme disease (LD) is the most common tick-borne disease caused by bacteria spirochetes of the Borrelia species, classified as Borrelia burgdorferi (Bb) strain [1,2]. Most cases of LD are reported in the northern hemisphere, mainly in the US and Europe [3–6]. Vector ticks are spirochetes of the genus Ixodes. An average LD incubation period takes from 3 to 30 days, although it might be prolonged up to 3 months. The typical clinical picture of LD may include skin, joints, heart, and/or nervous system symptoms, less often symptoms involving the eyes [7]. Depending on the symptoms and their time of onset, LD is classified in Lyme neuroborreliosis (LN) or Lyme arthritis (LA), and in early localized, early disseminated, or late disseminated infection. J. Clin. Med. 2019, 8, 929; doi:10.3390/jcm8070929 www.mdpi.com/journal/jcm J. Clin. Med. 2019, 8, 929 2 of 8 Symptoms may affect more than one organ, also including skin and eyes [8,9]. Lyme neuroborreliosis occurs most commonly as root pain, lymphocytic meningitis, cranial nerve palsies, especially involving nerve VII, or less frequently as peripheral nerve palsies [8,9]. LA usually affects large joints (e.g., the knees) and is usually asymmetrical. Arthritis can occur in other joints, including the shoulders, elbows, wrists, and ankles [9,10] and manifests with a slight swelling and pain [7–9]. The most common non-dental orofacial pain conditions are temporomandibular disorders (TMDs) [11]. Apart from pain, TMD signs include limitation of mouth opening and joint sounds. Similar symptoms can be caused also by other orofacial or systemic disorders, even including LD. There are limited studies regarding TMD involvement in patients with LD, hence the relationship between LD and TMD is still unclear. A temporomandibular joint involvement was frequently reported in early studies of LA [12,13], however it has been mentioned less often over the current years. In subsequent studies of Heir and Fein, Wolańska-Klimkiewicz et al., and te Veldhuis et al., an increase in reporting jaw-muscle symptoms in LD patients was noticed [14–16]. Based on previous research by Koutris et al. and Osiewicz et al., a differential diagnosis between TMD and comorbid diseases is implemented by the use of function-dependent tests. In particular, using static/dynamic tests may help reducing the false positives [17–19] as well as building a prediction model to assess which factors predict the presence of TMD symptoms in combination with comorbid disease, such as LD. This would be of help for the dentists to refer a patient to a proper specialist for a diagnosis and LD treatment. It would also help limiting delays in interventions and/or unnecessary treatment. Considering these drawbacks of previous studies and the scarceness of research on this topic, the aims of the present study were: 1. 2. 3. To assess the occurrence of TMD symptoms in patients with LD; To compare the occurrence of TMD symptoms in patients with LN and LA; To estimate the contribution of various factors that may predict TMDs in LD patients. 2. Materials and Methods 2.1. Participants The study was conducted at the Department of Infectious and Tropical Diseases of the University Hospital in Krakow, Poland, on 76 consecutive adult patients with LD seen between the years 2013 and 2016 (mean age 57.6 ± 14.6) and 54 healthy non-LD volunteers with a mean age of 56.4 ± 13.5 years. Volunteers of the control group were consecutively recruited from among the patients of the Dental Institute of the Jagiellonian University in Krakow, Poland. All subjects were aged over 18 years and gave informed consent. Based on a medical history and clinical examination, the presence of LD was excluded from the control group. The infection was confirmed by examining the presence of specific antibodies in serum and/or cerebrospinal fluid (CSF) using ELISA test (Biomedica, Vienna, Austria), and the borderline or positive results in serum were confirmed with the use of Western-blot test (recomBlot Borrelia, Microgen, London, UK). In the group of patients with suspected LN, lumbar puncture was performed, examining the level of cytosis, protein concentration, glucose, and chlorides [8,9]. The patients were divided into two groups: LA and LN. Diagnostic tests for LD were carried out by an infectious disease specialist (GB). The study was conducted according to the principles of the Helsinki Declaration and approved by the Bioethics Committee of the Jagiellonian University (No. KBET/200/B/2011). 2.2. Study Design During a dedicated session at the Department of Infectious and Tropical Diseases, a specialist in TMD (MO) performed an examination according to the validated Polish version of Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) guidelines [20–22]. All participants also completed the RDC/TMD history-taking questionnaire and the Axis II to assess potential predictors: J. Clin. Med. 2019, 8, 929 3 of 8 Sleep bruxism (SB), awake bruxism (AB), somatisation (SOM), depression (DEP), graded chronic pain scale (GCPS), other painful joints (OPJ), tinnitus, headaches or migraines (HM), education (high education or no high education), gender, and age. The examiner was trained in performing RDC/TMD clinical examination and dynamic/static tests by a calibrated examiner (FL) during a 3-year TMD and Orofacial Pain specialization program at the Department of Orofacial Pain and Dysfunction, Academic Centre for Dentistry Amsterdam (ACTA) [23]. Each patient had a panoramic radiograph provided by the Dental Institute of the Jagiellonian University in Krakow to exclude pain of possible dental origin. In the next phase, the RDC/TMD was used to establish possible function (i.e., non-pain) diagnoses of the masticatory system. IIA disc displacement with reduction: The disc is displaced from its position between the condyle and the fossa to an anterior and medial or lateral position, but reduces during full opening; IIB disc displacement without reduction with limited mouth opening: A condition in which the disc is displaced from its normal position between the condyle and the fossa to an anterior and medial or lateral position, associated with limited mandibular opening; IIC disc displacement without reduction, without limited opening: A condition in which the disc is displaced from its normal position between the condyle and the fossa to an anterior and medial or lateral position, not associated with limited opening; and IIIC osteoarthrosis: Coarse crepitus in the temporomandibular joint (TMJ) and/or tomograms showing pathology in the TMJ [20–22]. For pain diagnoses, function-dependent dynamic and static tests were performed, following the methods that were previously described by Visscher et al. and Osiewicz et al. [18,19,24]. Those tests aim to elicit function-dependent pain in the temporomandibular joints and/or masticatory muscles through loaded movements of the mandible and heavy static muscular effort, respectively, and are an expansion of the RDC/TMD protocol. Based upon the measurements derived from the clinical examination, the following TMD-pain diagnoses could be established: Mainly myogenous pain (MMP) and mainly arthogenous pain (MAP). 2.3. Data Analysis Categorical data were expressed as number and percentage, and comparisons between LD and controls and between LA and LN were performed using chi-square (χ2 ) or Fisher exact tests. The single logistic regression model was estimated for the different independent variables. Only those factors that were significant at p < 0.1 in the single-variable logistic regression analysis were included in the initial multiple variable regression model. Then, the variable with the weakest association was removed from the model. This procedure was repeated until all the variables that were retained in the model showed a p < 0.05. The odds ratios (OR) for MMP and/or MAP were assessed for each variable. OR values higher than 2 are commonly considered significant from a clinical viewpoint. Nagelkerke’s R-square was obtained as an estimation of the total variance explained by the predictor factors included in the model. Statistics were performed using StatsDirect, version 2.8.0 (StatsDirect Ltd., Cambridge, UK). 3. Results LD patients and control group did not differ significantly in the frequency of IIA diagnosis. Any subjects in the control group or in the LD patients group showed signs of IIB and IIC diagnoses. LD patients showed a significantly higher frequency (p < 0.001) of IIIC diagnosis than the control group. The frequency of MMP and/or MAP in LD patients (18.4%) was not significantly different in comparison to the control group (7.4%). The result was significant at the 10% level, but not at the 5% level (p = 0.059). The frequency of SB and AB in the two groups was not significantly different. In the LD group, moderate or severe SOM was shown in 73.7% of patients, whilst in the control group the frequency was 7.4% (p < 0.001). The frequency of high levels of chronic pain-related impairment (i.e., GCPS grades III or IV) was also significantly different between the LD patients and the control group at the level of p = 0.002. Chronic pain-related impairment in the LD group was shown in 15.8% of patients, whilst none J. Clin. Med. 2019, 8, 929 4 of 8 of the control participants showed this. There were significantly more patients with moderate and severe DEP (55.3%) in the LD group than in the control group (7.4%; p < 0.001). Similar differences were observed in the case of OPJ (LD group: 46%, control group: 14.8%), tinnitus (53.9% in the LD group and 3.7% in the control group), and HM (64.5% in the LD group and 11.1% in the control group) (Table 1). Table 1. Frequency of the Research Diagnostic Criteria of Temporomandibular Disorders (RDC/TMD)-based diagnosed of temporomandibular joint (TMJ) status, function-dependent tests, and of Axis II findings including sleep bruxism (SB) and awake bruxism (AB) in Lyme patients and control group. Lyme Patients IIA IIB/IIC IIIC MMP and/or MAP SB AB SOM GCPS DEP OPJ tinnitus HM 8 0 13 14 16 17 56 12 42 35 41 49 10.5% 0,0% 17.1% 18.4% 21% 22.4% 73.7% 15.8% 55.3% 46% 53.9% 64.5% p = Value Control 7 0 3 4 7 9 4 0 4 8 2 6 12.96% 0.0% 5.6% 7.4% 12% 16.7% 7.4% 0.0% 7.4% 14.8% 3.7% 11.1% p=1 p=1 p < 0.001 p = 0.059 * p = 0.234 p = 0.423 p < 0.001 p = 0.002 p < 0.001 p < 0.001 p < 0.001 p < 0.001 Disc displacement with reduction (IIA); disc displacement without reduction with limited mouth opening (IIB); disc displacement without reduction, without limited opening (IIC); osteoarthrosis (IIIC); mainly myogenous pain (MMP) and/or mainly arthogenous pain (MAP); sleep bruxism (SB); awake bruxism (AB); moderate/severe somatisation (SOM); high levels of chronic pain-related impairment (GCPS); moderate/severe depression (DEP); other painful joints (OPJ); tinnitus; headaches or migraines (HM).* Fisher exact test. In the LD group, half of the patients (N = 38) were diagnosed with LA and half with LN. The two groups did not significantly differ in the frequency of RDC/TMD-based function (non-pain) diagnoses, nor in that of the function-dependent pain diagnoses. The frequency of self-reported AB was significantly higher in the group of patients with LA (34.2%) than with LN (10.5%) (p = 0.013). Similar differences were observed for moderate and severe SOM, which was more frequent in patients with LA (84.21%) than with LN (63.16%) (p = 0.033). The frequency of moderate and severe DEP was also significantly higher in the LA group than in the LN group (65.8% vs. 44.7%). Similar differences (p = 0.010) were observed for OPJ (LA, 60.5%; LN, 31.6%) (Table 2). For the prediction of MMP and/or MAP in LD, the single regression analyses showed a possible association with age, SB, and AB. Two predictors (i.e., SB (p = 0.002) and AB (p = 0.017)) were statistically significant in the final multiple variable model that best predict the presence of positive outcomes of functional pain tests, viz., MMP and/or MAP in the LD group. Both SB and AB significantly increased the risk for MMP and/or MAP, with an odds ratio of 6.4 for AB and for 18.8 for SB (Table 3). J. Clin. Med. 2019, 8, 929 5 of 8 Table 2. Frequency of the RDC/TMD-based diagnosed of TMJ status, function-dependent tests and Axis II findings including SB and AB in Lyme arthritis (LA) and Lyme neuroborreliosis (LN) patients. LA (n = 38) LN (n = 38) p-Value IIA 4 10.5% 4 10.5% p=1 IIB 0 0.0% 0 0,0% p=1 IIC 0 0.0% 0 0,0% p=1 IIIC 9 23.7% 4 10.5% p = 0.128 MMP and/or MAP 8 21% 6 15.8% p = 0.554 SB 10 26.3% 6 15.8% p = 0.260 AB 13 34.2% 4 10.5% p = 0.013 SOM 32 84.2% 24 63.2% p = 0.033 GCPS 6 15.8% 6 15.8% p=1 DEP 25 65.8% 17 44.7% p = 0.050 OPJ 23 60.5% 12 31.6% p = 0.010 tinnitus 21 55.3% 20 52.6% p = 0.818 HM 27 71% 22 57.9% p = 0.231 Table 3. Single and multiple logistic regression models for the prediction of mainly myogenous pain (MMP) and/or mainly arthogenous pain (MAP) in Lyme disease (LD) patients. For each single regression, the number of cases included in the analysis is shown. Associations are expressed as odds ratio (OR) and 95% confidence interval (CI). For each removed predictor variable, the p-to-Exit is reported. Predictor Variable Gender female male Age Education SB AB SOM GCPS DEP OPJ Tinnitus HM no yes no yes no yes no yes no yes no yes no yes no yes no yes 35 41 76 55 21 60 16 59 17 20 56 64 12 34 42 41 35 35 41 27 49 Multiple Regression (N = 76) Single Regression Number p-Value OR p-Value p = 0.791 1.2 0.4 3.8 p = 0.790 OR p = 0.008 p = 0.166 1 2.4 0.9 0.7 1 7.9 p = 0.023 p = 0.172 p = 0.173 1 0.9 1.0 p < 0.001 23.3 5.6 97.8 p < 0.001 p ≤ 0.001 18.8 3.7 96 p < 0.001 12.2 3.2 45.6 p < 0.001 p = 0.027 6.4 1.2 33 p = 0.647 1.4 0.3 5.6 p = 0.064 p = 0.525 1.6 0.4 6.9 p = 0.535 p = 0.455 1.6 0.5 5.3 p = 0.449 p = 0.743 1.2 0.4 3.9 p = 0.743 p = 0.1371 0.4 0.1 1.3 p = 0.129 p = 0.2313 2.3 0.6 9.2 p = 0.208 95% CI p to Exit 95% CI 4. Discussion Findings of earlier studies suggest that dynamic/static tests should be used as a part of the routine TMD assessment [17–19]. An investigation by Osiewicz et al. in the population of patients with LD showed that 70% of them tested positive for RDC/TMD diagnosis of myofascial pain. Based on that, it might be suggested that palpation tests may be positive in TMD disorders of muscular origin in patients with other know conditions, such as LD, possibly due to the fact that the presence of comorbid conditions causes widespread muscle sensitization. Within these premises, the first aim of this study was to assess the occurrence of TMDs in patients with LD and to assess possible differences between J. Clin. Med. 2019, 8, 929 6 of 8 patients with LA and LN. Function (i.e., non-pain) diagnoses of the masticatory system were based on RDC/TMD, whist pain diagnoses were based on the function-dependent dynamic and static tests. The second aim was to estimate the contribution of various factors that might identify TMD in LD patients. The frequency of TMD was not significant in patients with LD in comparison with the control group as well as between LA and LN. It was only in the case of IIIC RDC/TMD diagnosis that Lyme patients showed a significantly higher frequency. A broadly accepted definition of LA states that an inflammation applies mostly to large joints [25], but a TMJ might be also inflamed. LD usually results in arthritis, but long lasting and untreated LA can cause a destruction of the joint, also known as osteoarthrosis. This investigation suggests that concurrent bruxism report may be the main factor to predict the presence of function-dependent TMD pain in patients with LD, to discriminate from those with Lyme-related pain. The risk for positive static/dynamic and/or palpation test in Lyme patients is much higher when the patients report sleep and/or awake bruxism. The role of bruxism in the etiology of TMDs is a much debated topic, because of the controversial relationship between self-reported bruxism and TMD symptoms as well as the difficulties to identify a dose–response relationship. Thus, it has become one of the most controversial issues within dental literature [26,27]. The absence of data on function-dependent TMD symptoms in LD makes it impossible to compare the present findings with the available literature. Earlier studies on TMD occurrence in patients with other primary conditions, such as irritable bowel syndrome (IBS) or Hashimoto thyroiditis, which usually showed a higher frequency of TMDs than in the general population, were based on palpation tests only [28,29]. Thus, such findings are potentially related to generalized hypersensitivity and not only to local musculoskeletal complaints [17–19,30]. There are several factors that might have affected the outcomes of this investigation. The main problem in this study was that the regression model included factors that were not assessed based on the best available methods. For instance, awake and sleep bruxism were just screened with the questions included in the history-taking questionnaire of the RDC/TMD. Future studies should assess AB by means of an Ecological Momentary Assessment, and SB by means of at least electromyography [31–33]. Another critical issue is that dynamic/static tests are not included in the standard protocols to diagnose TMD pain yet; they are only recommended as possible complimentary tools [34]. Thus, a suitable training in the specific techniques and in the verbal instructions for the patients is necessary. The adoption of the updated DC/TMD version plus the complimentary function-dependent tests is a recommended strategy to further increase the strength of similar data for cross-cultural comparison. As an additional finding, it is remarkable that the psychosocial status of patients with LD was compromised in comparison to the control group. However, function-dependent tests are not potentially related to a generalized hypersensitivity, so it can be hypothesized that such psychosocial status was related with the presence of LD itself. On the other hand, one of the strengths of this study is that for the first time, it assessed the frequency of TMD symptoms based on function-dependent static/dynamic tests. It also suggested that, even in the presence of concurrent conditions, the main factor that increases the risk for function-dependent TMD-pain diagnosis has to be searched in the complex spectrum of bruxism activities. The clinical significance of the study’s results confirms that dynamic/static tests should be used as a part of the routine TMD assessment. If the tests are negative, the patient should be referred to an infectious disease specialist to save valuable time spent on unnecessary treatment and to prevent delay of the appropriate intervention. 5. Conclusions With respect to the threefold aim, the results of this study suggest that the frequency of TMD was not significant in the patients with LD and that there are no differences between patients with LA or LN. This investigation suggests that the impact of bruxism to predict the presence of TMD pain in patients with LD is high. J. Clin. Med. 2019, 8, 929 7 of 8 Author Contributions: M.O. designed the study, performed the study, wrote, and revised the article. D.M. supervised and critically revised the article. G.B. carried out diagnostic tests for LD and critically revised the article. F.L., G.A., J.C., A.G., and J.P.-P. critically revised the article. Conflicts of Interest: The authors declare that there was no conflict of interest. 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Te Veldhuis, E.; Lobbezoo, F.; te Veldhuis, A.; Visscher, C.; Naeije, M.; van Selms, M. Residual orofacial complaints following Lyme neuroborreliosis: An unusual case of TMD. J. Craniomandib. Funct. 2011, 3, 221–228. Koutris, M.; Visscher, C.M.; Lobbezoo, F.; Naeije, M. Comorbidity negatively influences the outcomes of diagnostic tests for musculoskeletal pain in the orofacial region. Pain 2013, 154, 927–932. [CrossRef] Osiewicz, M.A.; Manfredini, D.; Loster, B.W.; van Selms, M.K.A.; Lobbezoo, F. Comparison of the outcomes of dynamic/static tests and palpation tests in TMD-pain patients. J. Oral Rehabil. 2018, 45, 185–190. [CrossRef] Osiewicz, M.; Manfredini, D.; Biesiada, G.; Czepiel, J.; Garlicki, A.; Pytko-Polończyk, J.; Lobbezoo, F. Differences between palpation and static/dynamic tests to diagnose painful temporomandibular disorders in patients with Lyme disease. Clin. Oral Investig. 2019, 34, 74–81. [CrossRef] Dworkin, S.F.; LeResche, L. Research diagnostic criteria for temporomandibular disorders: Review, criteria, examinations and specifications, critique. J. Craniomandib. Disord. 1992, 6, 301–355. J. Clin. Med. 2019, 8, 929 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 8 of 8 Osiewicz, M.A.; Lobbezoo, F.; Loster, B.W.; Wilkosz, M.; Naeije, M.; Ohrbach, R. Badawcze kryteria diagnostyczne zaburzeń czynnościowych układu ruchowego narzadu ˛ żucia BKD/ZCURNŻ—Polska wersja dwuosiowego systemu diagnostycznego ZCURNŻ. Protet Stomatol 2010, 60, 433–444. Osiewicz, M.; Lobbezoo, F.; Loster, B.; Wilkosz, M.; Naeije, M.; Ohrbach, R. Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD)—The Polish version of a dual-axis system for the diagnosis of TMD. RDC/TMD Form. J. Stomatol. Czasopismo Stomatol. 2013, 66, 576–649. [CrossRef] Lobbezoo, F.; van der Zaag, J.; Visscher, C.M.; Naeije, M. Oral kinesiology. A new postgraduate programme in the Netherlands. J. Oral Rehabil. 2004, 31, 192–198. [CrossRef] Visscher, C.M.; Lobbezoo, F.; Naeije, M. A reliability study of dynamic and static pain tests in temporomandibular disorder patients. J. Orofac. Pain 2007, 21, 39–45. [PubMed] Arvikar, S.; Steere, A. Diagnosis and Treatment of Lyme Arthritis. Infect. Dis. Clin. N. Am. 2015, 29, 269–280. [CrossRef] [PubMed] Manfredini, D.; Lobbezoo, F. Relationship between bruxism and temporomandibular disorders: A systematic review of literature from 1998 to 2008. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 2010, 109, e26–e50. [CrossRef] [PubMed] Muzalev, K.; Visscher, C.M.; Koutris, M.; Lobbezoo, F. Long-term variability of sleep bruxism and psychological stress in patients with jaw-muscle pain: Report of two longitudinal clinical cases. J. Oral Rehabil. 2018, 45, 104–109. [CrossRef] [PubMed] Gallotta, S.; Bruno, V.; Catapano, S.; Mobilio, N.; Ciacci, C.; Iovino, P. High risk of temporomandibular disorder in irritable bowel syndrome: Is there a correlation with greater illness severity? World, J. Gastroenterol. 2017, 23, 103–109. [CrossRef] [PubMed] Grozdinska, A.; Hofmann, E.; Schmid, M.; Hirschfelder, U. Prevalence of temporomandibular disorders in patients with Hashimoto thyroiditis. J. Orofac. Orthop. 2018, 79, 277–288. [CrossRef] Visscher, C.M.; Naeije, M.; De Laat, A.; Michelotti, A.; Nilner, M.; Craane, B.; Ekberg, E.; Farella, M.; Lobbezoo, F. Diagnostic accuracy of temporomandibular disorder pain tests: A multicenter study. J. Orofac. Pain 2009, 23, 108–114. Lobbezoo, F.; Ahlberg, J.; Raphael, K.G.; Wetselaar, P.; Glaros, A.G.; Kato, T.; Santiago, V.; Winocur, E.; De Laat, A.; De Leeuw, R.; et al. International consensus on the assessment of bruxism: Report of a work in progress. J. Oral Rehabil. 2018, 45, 837–844. [CrossRef] [PubMed] Zani, A.; Lobbezoo, F.; Bracci, A.; Ahlberg, J.; Manfredini, D. Ecological Momentary Assessment and Intervention Principles for the Study of Awake Bruxism Behaviors, Part 1: General Principles and Preliminary Data on Healthy Young Italian Adults. Front. Neurol. 2019, 10, 169. [CrossRef] Osiewicz, M.A.; Lobbezoo, F.; Bracci, A.; Ahlberg, J.; Pytko-Polończyk, J.; Manfredini, D. Ecological Momentary Assessment and Intervention Principles for the Study of Awake Bruxism Behaviors, Part 2: Development of a Smartphone Application for a Multicenter Investigation and Chronological Translation for the Polish Version. Front. Neurol. 2019, 10, 170. [CrossRef] [PubMed] Schiffman, E.; Ohrbach, R.; Truelove, E.; Look, J.; Anderson, G.; Goulet, J.-P.; List, T.; Svensson, P.; Gonzalez, Y.; Lobbezoo, F.; et al. Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) for Clinical and Research Applications: Recommendations of the International RDC/TMD Consortium Network* and Orofacial Pain Special Interest Group. J. Oral Facial Pain Headache 2014, 28, 6–27. [CrossRef] [PubMed] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). 

W1598156814.txt

We are IntechOpen, the world’s leading publisher of Open Access books Built by scientists, for scientists 6,900 186,000 200M Open access books available International authors and editors Downloads Our authors are among the 154 TOP 1% 12.2% Countries delivered to most cited scientists Contributors from top 500 universities Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) Interested in publishing with us? Contact book.department@intechopen.com Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com 1 Management of Women with Clomifene Citrate Resistant Polycystic Ovary Syndrome – An Evidence Based Approach Hatem Abu Hashim Department of Obstetrics & Gynecology, Faculty of Medicine, Mansoura University, Mansoura, Egypt 1. Introduction World Health Organisation (WHO) type II anovulation is defined as normogonadotrophic normoestrogenic anovulation and occurs in approximately 85% of anovulatory patients. Polycystic ovary syndrome (PCOS) is the most common form of WHO type II anovulatory infertility and is associated with hyperandrogenemia (1,2). Moreover, PCOS is the most common endocrine abnormality in reproductive age women. The prevalence of PCOS is traditionally estimated at 4% to 8% from studies performed in Greece, Spain and the USA (3-6). The prevalence of PCOS has increased with the use of different diagnostic criteria and has recently been shown to be 11.9 ± 2.4% -17.8 ± 2.8 in the first community-based prevalence study based on the current Rotterdam diagnostic criteria compared with 10.2 ± 2.2% -12.0 ± 2.4% and 8.7 ± 2.0% using National Institutes of Health criteria and Androgen Excess Society recommendations respectively (7). Importantly, 70% of women in this recent study were undiagnosed (7). Clomiphene citrate (CC) is still holding its place as the first-line therapy for ovulation induction in these patients (2,8,9). CC contains an unequal mixture of two isomers as their citrate salts, enclomiphene and zuclomiphene. Zuclomiphene is much the more potent of the two for induction of ovulation, accounts for 38% of the total drug content of one tablet and has a much longer half-life than enclomiphene, being detectable in plasma 1 month following its administration (10). CC is capable of inducing a discharge of FSH from the anterior pituitary and this is often enough to reset the cycle of events leading to ovulation into motion. This is achieved indirectly, through the action of CC, a non-steroidal compound closely resembling an estrogen, in blocking hypothalamic estrogen receptors, signalling a lack of circulating estrogen to the hypothalamus and inducing a change in the pattern of pulsatile release of GnRH(10). Standard practice is to administer CC for 5 days from the second or third day of the menstrual cycle, starting with 50mg/day and increasing to 250mg/day (10). However managed care studies have shown that the most effective dosage is 100–150mg/day and over 75% of ovulations occur within these dosages (11). After six to nine cycles of treatment with CC cumulative pregnancy rates reach 70–75% (11). Life table analysis of the most reliable studies indicated a conception rate up to 22% per cycle in women ovulating on CC (8). In a large randomized trial, Legro et al., 2007 (12) compared the www.intechopen.com 2 Polycystic Ovary Syndrome effects of CC, metformin and combination therapy in 626 infertile women with PCOS. They reported an ovulation and clinical pregnancy rates per woman of 75.1% and 23.9% respectively, after CC treatment up to 150mg/day. Clomiphene resistance defined as failure to ovulate after receiving 150 mg of CC daily for 5 days per cycle, for at least three cycles, is common and occurs in approximately 15 to 40% in women with PCOS (2, 13). Insulin resistance, hyperandrogenemia, and obesity represent the major factors involved in CC resistance; avert the ovaries from responding to raised endogenous FSH levels following CC therapy (14-16). Moreover, a genetic predisposition was suggested (17). The purpose of this chapter is to review the evidence based treatment strategies for ovulation induction in anovulatory PCOS patients with known CC resistance, both the traditional and new ones. The traditional options include gonadotrophins and surgery (laparoscopic ovarian drilling). New strategies as insulin-sensitizing drugs, aromatase Inhibitors, oral contraceptives, dexamethasone, N-acetyl-cysteine…etc. Moreover, optimizing the body mass index (BMI) firstly before commencing therapy is an important issue to improve the treatment outcome in obese anovulatory women with PCOS. In vitro fertilization (IVF) is the recommended line of treatment after failure of these strategies; however, it is outside the scope of this chapter. Finally an algorithm will be provided to facilitate management of this important clinical issue. 2. Weight loss and lifestyle modifications Obesity is strongly associated with PCOS and may be present in up to 50% of cases (18-22). Obese women with PCOS are more likely than thin women with PCOS to suffer from anovulation (18). This effect on ovulation may be secondary to insulin resistance, which in turn results in hyperinsulinemia and stimulation of excess androgen production from the ovaries (22). Lifestyle modification is the first line treatment in an evidence based approach for the management of the majority of PCOS women who are overweight (8,9,13, 23-25). The NICE, 2004 (13) recommended weight loss for anovulatory PCOS women who have a BMI > 29 kg/m2 before starting ovulation induction therapy. In these women, weight loss of even 5% to 10% of body weight often restores ovulatory cycles (9, 19, 21). Studies also showed that overweight women are less likely to respond to pharmacologic ovulation induction methods. In a cohort of 270 women, with PCOS who received either CC or gonadotrophins for ovulation induction, almost 80% with a BMI of 18–24 kg/m2 ovulated at 6 months compared with only 12% of women with a BMI≥35 kg/m2 (18). Moreover, overweight women require higher doses of CC and gonadotrophins (19). The current recommendation is to reduce weight gradually to increase the chances of maintaining the weight loss (9). Preferential diet composition has been evaluated in 2 small studies (26, 27). These studies compared a high carbohydrate (55%), low protein (15%) hypocaloric diet with a low carbohydrate (40%), high protein (30%) hypocaloric diet and found similar weight loss and decrease in circulating androgen and insulin levels. Routine exercise is also very important in the reproductive health of PCOS women. Exercise increases insulin sensitivity and helps achieve and maintain weight loss (9, 25). Incorporating simple moderate physical activity including structured exercise (at least 30 min/ day) and incidental exercise increases weight loss and improves clinical outcomes in PCOS, compared to diet alone (28). Also, a recent study reported that a 6-week intervention www.intechopen.com Management of Women with Clomifene Citrate Resistant Polycystic Ovary Syndrome – An Evidence Based Approach 3 of structured exercise training and a hypocaloric diet was effective in increasing the probability of ovulation under CC treatment in overweight and obese CC-resistant PCOS patients (29). Other lifestyle factors such as excessive caffeine intake, alcohol consumption, and smoking should also be addressed (13,20). Otta et al., 2010 (30) in a randomized, double-blind, and placebo control trial compared lifestyle modification and 1500 mg of metformin or placebo for 4 months in 30 women with insulin resistance PCOS. They reported that metformin has an additive effect to diet and exercise to improve parameters of hyperandrogenism and insulin resistance. However, a small decrease in body weight through lifestyle changes could be enough to improve menstrual cycles in these women. Karimzadeh & Javedani, 2010 (31)in another randomized double-blind study compared lifestyle modification with medical treatment plans such as CC, metformin, and CC with metformin in 343 overweight infertile women with PCOS. They showed that metformin or metformin with CC does not cause a significant weight loss or an improvement in the endocrine status of PCOS women. However, lifestyle modification to reduce waist circumference and body weight could improve their menstrual cycles, hormonal status and was an effective treatment for ovulation induction in those patients with an ovulation and pregnancy rates of 66.6% and 20% respectively. In morbidly obese women, the PCOS phenotype appears to be very frequent (32). Importantly, this disorder has been found to improve markedly after sustained weight loss following bariatric surgery (33). Anti-obesity pharmacological agents have been used in obese women with PCOS. Both orlistat, which blocks intestinal absorption of fat (34), and sibutramine, an appetite suppressant (35), have displayed a weight loss-independent effect on androgens and insulin resistance. It should be noted that these treatments should not be considered as first-line therapy for obesity in women with PCOS (8). 3. Gonadotrophins Ovulation induction for women with anovulatory PCOS using gonadotrophin therapy is based on the physiological concept that initiation and maintenance of follicle growth may be achieved by a transient increase in FSH above a threshold dose for sufficient duration to generate a limited number of developing follicles (8). Traditionally, Ovulation induction with gonadotrophins has been used as a second line treatment for CC-resistant PCOS women, however it is expensive, requires extensive monitoring and associated with significantly increased risk for ovarian hyperstimulation syndrome (OHSS) and multiple pregnancy (8, 9, 13, 36-38). Furthermore, a significant and consistent relationship between PCOS and OHSS was reported in a systematic review (39). The high sensitivity of the PCOS to gonadotrophic stimulation is probably related to the fact that they contain twice the number of available follicle-stimulating hormone (FSH)-sensitive antral follicles in their cohort than the normal ovary (40). A meta-analysis concluded that the outcomes of treatment achieved with hMG and with FSH alone in infertile patients with PCOS were similar except for a reduction in the risk of OHSS with the urinary FSH (uFSH) (41). A lowdose, step-up gonadotrophin therapy should be preferred to the now outdated conventional protocol for patients with PCOS and the strong justification seems to be; the achievement of high rate of mono-follicular development which is ~69% (54–88%) (36,42) with nearly complete elimination of OHSS (0–2.4%) and a multiple pregnancy rate of ~6% (36,43). The recommended approach is to begin with a low dose of gonadotrophin, typically 37.5– 75 www.intechopen.com 4 Polycystic Ovary Syndrome IU/day, increasing after 7 days or more if no follicle >10 mm has yet emerged, in small increments, at intervals, until evidence of progressive follicular development is observed. The maximum required daily dose of FSH/hMG seldom exceeds 225 IU/day (38, 44). There is no evidence of a difference between recombinant FSH (rFSH) and uFSH for ovulation induction in CC- resistant PCOS women (45,46). In addition, a randomised trial (RCT) of highly purified uFSH (HP-uFSH) versus rFSH found that the former was non-inferior compared with the latter with respect to ovulation rate (85.2% versus 90.9%) in anovulatory WHO Group II women who failed to ovulate or conceive on CC (47) . 4. Laparoscopic Ovarian Diathermy (LOD) Laparoscopic ovarian diathermy (LOD) is currently accepted as a successful second line treatment for ovulation induction in CC-resistant PCOS being as effective as gonadotrophin treatment and is not associated with an increased risk of multiple pregnancy or OHSS (8, 9,13, 48-51). Bayram et al., 2004 (50) in a RCT compared LOD with rFSH in 168 CC-resistant PCOS women. They reported an ovulation rate of 70% and 69% per cycle and pregnancy and live-birth rates 37%, 75% and 34%, 60% of patients respectively following LOD and FSH therapy. In patients remaining anovulatory 8 weeks after LOD or those who subsequently became anovulatory, adjuvant therapy with CC or gonadotrophins was required to achieve equivalent pregnancy and live-birth rates (50). A Cochrane review found no difference in the rates of miscarriage, ongoing pregnancy or live birth between LOD and gonadotrophins. Multiple pregnancy rates were significantly lower with LOD than with gonadotrophins (1% versus 16%; OR 0.13, 95% CI 0.03 to 0.52) (49). A recent study concluded that LOD for women with CC-resistant PCOS is as effective as ovulation induction with rFSH treatment in terms of live births, but reduces the need for ovulation induction or ART in a significantly higher proportion of women and increases the chance for a second child (52). The main shortcomings of LOD are the need for general anesthesia and the risk of postoperative adhesions (53, 54). The claim that it might affect the ovarian reserve is not more than a theoretical concern since a recent report concluded that LOD, when applied properly, does not seem to compromise the ovarian reserve in PCOS women (55). Moreover, an economic evaluation has shown that the cost of a live birth after LOD is approximately one-third lower than the equivalent cost of gonadotrophin treatment (56). The most commonly used energy for LOD is electrocautery. It has been reported that the clinical and endocrine response to LOD is governed by a dose response relationship. Four punctures per ovary using a power setting of 30 W applied for 5s per puncture (i.e. 600 J per ovary) are sufficient to produce an optimal response (67% spontaneous ovulation rate and 67% conception rate). Reducing the thermal energy below that level reduces the chances of spontaneous ovulation and conception (57). Also, different studies argued for unilateral LOD being equally efficacious as bilateral drilling in inducing ovulation and achieving pregnancy in CC resistant PCOS patients and may be regarded as a suitable option with the potential advantage of decreasing the chances of adhesion formation (58-60). Although it remains unclear as to how LOD induces ovulation, a potential mechanism is that LOD drains the ovarian follicles containing a high concentration of androgens and inhibin, which causes the reduction of blood androgens and blood inhibin resulting in an increase of FSH and recovery of the ovulation function (51,53, 61,62). Surgery may also provoke an increased blood flow to the ovary, allowing increased delivery of www.intechopen.com Management of Women with Clomifene Citrate Resistant Polycystic Ovary Syndrome – An Evidence Based Approach 5 gonadotrophins (53, 62). Women with marked obesity (BMI >35 kg/m2), marked hyperandrogenism (serum testosterone concentration >4.5 nmol/l, free androgen index (FAI) >15) and/or long duration of infertility (>3 years) seem to be poor responders to LOD. On the other hand, high LH levels >10 IU/l in LOD responders appear to predict higher probability of pregnancy (63). van Wely et al., 2005 (64) reported that women who had an age at menarche < 13years, an LH/FSH ratio < 2 and a glucose level < 4.5 mmol/l, were more likely to remain anovulatory following LOD. Restoration of consecutive spontaneous ovulations after LOD in some CC-resistant PCOS patients is one of the most important advantages of this approach (65). Another potential advantage is the increased responsiveness of the ovary to oral ovulation induction agents following the procedure. In a recent study, we evaluated whether LOD in CC-resistant PCOS patients led to the restoration of CC-sensitivity. LOD was performed in 234 CCresistant PCOS patients. In 150 patients ovulation occurred. The remaining 84 aonvulatory patients were again treated with CC. Ovulation occurred in 30 /84 patients (35.7%), meanwhile, pregnancy occurred in 13/ 84 patients (15.5%). Hyperandrogenism and insulin resistance were negative predictors (66). 5. Insulin-sensitizing drugs Approximately 50%-70% of all women with PCOS have some degree of insulin resistance (67). Hyperinsulinemia probably contributes to the hyperandrogenism which is responsible for the signs and symptoms of PCOS (67). Metformin, a biguanide, is now the most widely insulin sensitizer used for ovulation induction in women with PCOS. In these women, it appears to affect ovarian function in a dual mode, through the alleviation of insulin excess acting upon the ovary and through direct ovarian effects. Being an insulin sensitizer, it reduces insulin secretion and, consequently, lowers circulating total and free androgen levels with a resulting improvement of the clinical sequelae of hyperandrogenism. Importantly, it also seems to have a direct action on ovarian theca cells to decrease androgen production (68). A recent meta-analysis of RCTs showd no significant difference in effectiveness of metformin versus CC as a first-line treatment for ovulation induction in non-obese women with anovulatory PCOS (69). Also a recent Cochrane review reported that metformin is still of benefit in improving clinical pregnancy and ovulation rates. However, there is no evidence that it improves live birth rates whether it is used alone or in combination with CC, or when compared with CC. Therefore, the use of metformin as a first-line treatment in improving reproductive outcomes in women with PCOS appears to be limited (70). Many investigators have demonstrated an improvement in insulin sensitivity and a significant decrease in serum insulin and free testosterone levels after long term treatment with metformin for 5–8 weeks (71-73). Creanga et al., 2008 (74) in a meta-analysis, confirmed that metformin in combination with CC increased the likelihood of ovulation [OR 4.39, 95% CI 1.94–9.96, number - needed- to-treat (NNT) 3.7] and pregnancy (OR 2.67, 95% CI 1.45– 4.94, NNT 4.6) in comparison with CC alone, especially in CC-resistant and obese PCOS patients. Actually, different mechanisms explaining why metformin therapy would facilitate ovulation induction by CC in CC- resistant PCOS patients have been proposed entailing; an intrinsic alteration of follicle steroidogenesis through the IGF-I pathway in granulosa cells (73); direct inhibition of androgen production in ovarian thecal cells (75); reduction in the www.intechopen.com 6 Polycystic Ovary Syndrome adrenal steroidogenesis response to ACTH (76) and recently its central action on the pituitary gland with an LH lowering and prolactin effects in the PCOS women (77). There are unpleasant gastrointestinal side effects including nausea, vomiting, bloating, cramps and diarrhoea. Rare complication includes lactic acidosis. Metformin has been used in increasing doses from 500 to 1500 mg daily for the induction of ovulation in women with PCOS (9). Recently, the efficacy of the combination of metformin and CC versus other traditional options including gonadotrophins and LOD for treatment of CC-resistant infertile PCOS patients has been reported. Two RCTs compared the combination of metformin and CC with LOD, showing that both are effective approaches to treat CC- resistant infertile PCOS patients (78, 79). In fifty primary infertile patients with CC- resistant PCOS, Palombo et al., 2010 (78) found no significant difference between the 2 groups in pregnancy and live-birth rates per cycle (13.1% vs.16.3% and 11.2% vs. 14.1% respectively). However, the ovulation rate per cycle was significantly lower in LOD group than in Metformin/CC group (56.5% vs. 72.0%). On the other hand, in a well designed adequately powered RCT comprised of 282 anovulatory women with CC-resistant PCOS, we reported no significant difference between the 2 groups in ovulation and pregnancy rates per cycle (67% vs. 68.2% and 15.4% vs. 17% respectively). However, a significant difference regarding midcycle endometrial thickness was found (9.2 ± 1.2 mm vs. 7.6 ± 1.1 mm, in Metformin/CC and LOD groups respectively) (79). George et al., 2003 (80) in a small trial of limited power compared sequential treatment of metformin and CC with conventional hMG protocol in 60 CC-resistant PCOS patients. In this trial, metformin alone was given as a single pretreatment for 6 months, followed by ovulation induction with CC. There was no significant difference in pregnancy rates between the two groups (16.7 vs. 23.3%). However, in the metformin group, significant improvements in menstrual function and ovulation rate of 46.7% with a significant decrease in fasting insulin levels were reported. The ovulation rate in hMG group was 43.3%, with a high drop-out rate. Recently, in a well designed adequately powered RCT we compared the effects of combined metformin–CC with HP-uFSH using low-dose, step-up regimen for three cycles in 153 anovulatory women with CC-resistant PCOS (81). Actually, combined metformin-CC therapy was not expected to be more effective than gonadotrophins, however, it did result in modest ovulation and pregnancy rates. Ovulation and pregnancy rates per cycle were 62% vs. 83.8% and 11.2% vs. 21.5% in combined metformin- CC group and HP-uFSH groups respectively. HP-uFSH administration had good results, but, the lowdose, step-up regimen requires extensive monitoring and expertise, and has high costs. Accordingly, it is logical to offer combined metformin- CC therapy first in the step-wise treatment protocol for CC-resistant PCOS patients before resorting to more expensive alternatives especially in developing communities where economic aspects of therapy are important (81). The safety of metformin has sparked a heated debate. Recent evidence that metformin is probably safe during the first trimester of pregnancy and beyond is accumulating (82-85). Moreover, a recent meta-analysis found no effect of pregestational metformin administration on abortion risk in PCOS patients (86). Other insulin sensitizers from the thiazolidenediones family, namely rosiglitazone, have been used effectively in CC-resistant PCOS patients. In a RCT, the combination of rosiglitazone and CC was reported to be more effective than metformin and CC in terms of ovulation rate (64.3 vs. 36.4%, respectively); whereas no statistical significance was observed in pregnancy rate (50 vs. 38.5%) (87). Also, a recent RCT reported no significant difference between combined treatment with www.intechopen.com Management of Women with Clomifene Citrate Resistant Polycystic Ovary Syndrome – An Evidence Based Approach 7 rosiglitazone and CC vs. LOD in 43 CC-resistant PCOS patients in terms of biochemical response, ovulation rate (80.8 vs. 81.5%) and pregnancy rate (50 vs. 42.8%) (88). A retrospective analysis investigated various clinical, biochemical, and ultrasonographic factors that determine clinical response to rosiglitazone as a first-line therapy in a series of PCOS women with newly diagnosed CC-resistance. It showed that marked obesity, marked hyperandrogenism, and long duration of infertility were predictors of resistance to rosiglitazone therapy (89). 6. Third-generation aromatase inhibitors Third-generation aromatase inhibitors (anastrozole, letrozole, exemestane) are approved adjuvants for treatment of estrogen-receptor–positive breast cancer (90) that were first used in ovulation induction in anovulatory women in 2001 (91). Evidence suggests that nonsteroidal aromatase inhibitors (AIs), specifically letrozole and anastrozole, have ovulation-inducing effects by inhibiting androgen-to-estrogen conversion. Centrally, this effect releases the hypothalamic/pituitary axis from estrogenic negative feedback, increases gonadotrophin secretion, and results in stimulation of ovarian follicle maturity. Moreover, peripherally, AIs may increase follicular sensitivity to FSH (92). AIs have relatively short half-lives (~2 days) compared with CC (~2 weeks) so estrogen target tissues (e.g., endometrium and cervix ) are spared adverse effects. Because of these mechanisms, it was postulated that AIs may have superior ovulation induction properties in terms of follicular growth and endometrium development, which is important for embryo implantation (92). Recent studies showed that letrozole has better ovulation and pregnancy rates in comparison to CC and placebo in patients with CC- resistant PCOS (93-96). There are 2 prospective studies in the literature comparing the two commercially available third generation AIs, letrozole and anastrozole in CC-resistant infertile women with PCOS. AlOmari et al., 2004 (97) studied 40 cases who were considered CC- resistant if failed ovulation after 200 mg CC daily for 5 days or were ovulatory with an endometrium thickness less than 5 mm. Ovulation and pregnancy rates per cycle were significantly higher with letrozole compared with anastrozole (84.4% vs. 60% and 18.8% vs. 9.7%, respectively). Endometrium thickness was significantly greater for letrozole compared with anastrozole (8.16 ± 1.32 vs. 6.53 ± 1.55 mm). Multiple pregnancies did not occur. In this small trial, PCOS diagnostic criteria were not stated. Additionally, the dose of CC used to define resistance was very high, possibly suggesting an extremely refractory population. Importantly, a larger RCT compared the efficacy of letrozole and anastrozole in 220 CC–resistant women with PCOS diagnosed with Rotterdam criteria. More growing and mature follicles and greater endometrial thickness in patients receiving anastrozole were demonstrated; however, no significant advantage for anastrozole over letrozole with regard to ovulation, pregnancy or miscarriage rates was observed (63.4 vs.62% and 15.1vs. 12.2% and 9.5vs. 11.1% respectively). Two twin pregnancies occurred with letrozole, while none occurred with anastrozole (98). In the above mentioned 2 studies, a short course (5 days) of letrozole was used. However, a long letrozole protocol (10 days) was also proposed, with proved advantages in terms of more mature follicles and subsequently more pregnancies (99). One small trial of limited power compared combined metformin–letrozole vs. metformin– CC in 60 CC-resistant PCOS patients reported that combined metformin–letrozole was www.intechopen.com 8 Polycystic Ovary Syndrome associated with significantly more endometrial thickness, E2 levels and full-term pregnancy rate. However, no statistically significant difference was found between the two groups as regards the mean number of mature follicles, ovulation and pregnancy rates. The authors admitted that combined metformin– letrozole is better than letrozole alone, particularly in overweight women and asked for further studies to confirm their hypothesis (100). Recently, in a well designed adequately powered RCT, we compared the effects of letrozole monotherapy (2.5 mg daily for 5 days from D3-7 of the cycle) with combined metformin–CC in 250 anovulatory women (582 cycles) with CC resistant PCOS. Our findings suggested that letrozole monotherapy and combined metformin-CC were equally effective for inducing ovulation and achieving pregnancy in patients with CC-resistant PCOS (64.9% vs.69.6% and 14.7% vs. 14.4% respectively). The total number of follicles was significantly more in the combined metformin–CC group (4.4 ± 0.4 vs. 6.8 ± 0.3). A non significant increase in endometrial thickness on the day of hCG administration was observed in the letrozole group (9.5 ± 0.2 mm vs. 9.1 ± 0.1 mm). Since letrozole was well tolerated, it is considered as an acceptable alternative if CC-resistant PCOS patients cannot tolerate long-term metformin pretreatment (101). More recently, the efficacy of the AIs vs. other traditional options including gonadotrophins and LOD for treatment of CC-resistant infertile PCOS patients has been reported. 2 RCTs compared the effect of letrozole (2.5mg and 5 mg respectively from day 3 to day 7 of menses for 6 consecutive cycles) with LOD for ovulation induction in CC resistant women with PCOS. Both trials reported that letrozole and LOD are equally effective for inducing ovulation and achieving pregnancy in these patients. Moreover, women in the letrozole group had a significantly thicker endometrium than those in the LOD group. In view of the invasiveness and cost of surgery, it seems plausible that letrozole therapy should be tried first for most of those women before shifting to LOD (102,103). A recent large randomized trial by Ganesh et al., 2009 (104) compared the efficacy of letrozole with that of rFSH and CC/rFSH for ovarian stimulation in IUI cycles in 1387 PCOS women after CC failure. They reported an ovulation rate of 79.30% in letrozole group vs. 56.95% and 89.89% in other groups respectively and pregnancy rate of 23.39% in letrozole group vs. 14.35% and 17.92% in other groups respectively. However, they included not only CC-resistant PCOS patients but also those who failed to conceive with100 mg/day CC for 6 cycles despite ovulating and those who showed poor endometrial development i.e. endometrial thickness < 7 mm on the day of hCG administration. Letrozole was evaluated in 44 women with CC–resistant PCOS and both responders and nonresponders were characterized. PCOS was diagnosed by Rotterdam criteria; CCresistance was defined as failure to ovulate after 6 cycles of 150 mg CC /day for 5 days. Whereas response to CC is less likely with elevated BMI, amenorrhea, and increased age, significant differences between letrozole responders and nonresponders were not noted for any evaluated measure. This apparent lack of predictive factors for letrozole suggests utility in CC–resistant patients since its efficacy is not limited to specific patient characteristics (105). The safety of letrozole has elaborated a vivid discussion. Preliminary data by Biljan et al., 2005 (106) suggested an increased risk of congenital anomalies in letrozole treated babies, whereas recent data from retrospective and prospective trials (107,108) have contested these initial findings and supported the safety of letrozole compared to traditional ovulation induction treatment. www.intechopen.com Management of Women with Clomifene Citrate Resistant Polycystic Ovary Syndrome – An Evidence Based Approach 9 7. Oral contraceptives Branigan & Estes., 2003 (109) in a RCT showed that the suppression of the hypothalamic pituitary- ovarian axis for 2 months with combined oral contraceptives (COC) (0.03 mg of ethinyl estradiol and 0.15 mg of desogestrel) followed by CC, at dosage of 100 mg/day on days fifth to ninth of the cycle, improved ovulation and pregnancy rates in CC resistant women in comparison with repeated cycles of CC alone. Oral contraceptive administration showed to reduce serum LH, estradiol and androgen levels. These hormonal changes, especially the reduced androgenic milieu, could act improving the ovarian microenvironment, and thus the ovarian response to CC. Kriplani et al., 2010 (110) in a RCT reported that in women with PCOS, a drospirenone containing COC has better outcome in terms of persistent regular cycles, antiandrogenic effect,fall in BMI and BP, better lipid profile, favorable glycemic and hormonal profile than desogestrel-containing COC. 8. N-acetyl-cysteine N-acetyl-cysteine (NAC) is a mucolytic drug. Fulghesu et al., 2002 (111) demonstrated that long term NAC treatment (1.8 g/d for 5–6 weeks) was associated with significant increase in insulin sensitivity and reduction in insulin levels, testosterone and FAI in hyperinsulinemic PCOS. Rizk et al., 2005 (112) showed that the combination of NAC (1.2 g/d) with CC (100 mg/d) for only 5 days significantly increased both ovulation and pregnancy rates in obese women with CC-resistant PCOS compared with placebo (49.3% vs. 1.3% and 21.3% vs. 0, respectively). Actually, these results supporting the shorter duration (5 days only) of NAC administration in CC- resistant PCOS women have not been replicated by other trials. Recently, in a well designed adequately powered RCT, we reported that the efficacy of metformin–CC combination therapy is higher than that of NAC – CC for inducing ovulation and achieving pregnancy among CC-resistant PCOS patients (113). In our study, the dose and duration of NAC were chosen based on that published by Fulghesu et al., 2002(111). Over a 3-month follow-up period, women in metformin-CC group had significantly higher ovulation and pregnancy rates compared with women in NAC-CC group (69.1% vs. 20.0% and 22.7% vs. 5.3%, respectively). Moreover, the level of serum estrogen, the endometrial thickness on the day of hCG administration and the midluteal serum progesterone level were all significantly higher for women in metformin-CC group than other group. Additionally, a lower miscarriage rate was observed among women in metformin-CC group (113). 9. Dexamethasone therapy Dexamethasone therapy during the follicular phase has been described without any side effects or serious events (114). Parsanezhad et al., 2002 (115) in a double-blind RCT, showed the safety and the efficacy of a high-dose short course of dexamethasone for inducing ovulation in 230 CC-resistant patients with PCOS and normal DHEAS levels. They reported significantly higher ovulation and pregnancy rates in those who received 200mg of CC (days 5–9) and 2mg of dexamethasone (days 5–14) compared with CC alone (88% vs. 20% and 40.5 vs. 4.2% respectively). In these patients, dexamethasone reduced circulating DHEAS, T, and LH levels and the LH/FSH ratio after 2 weeks of treatment (115). These results were further confirmed in another RCT (116). www.intechopen.com 10 Polycystic Ovary Syndrome 10. Bromocriptine Currently, evidence suggests that PCOS and hyperprolactinaemia are two distinct entities without a patho-physiological link (117-119). Bromocriptine administration provided no benefit in CC-resistant PCOS patients with normal prolactin levels, receiving 150mg CC (days 5–9) and bromocriptin continuously administrated at a dosage of 7.5 mg daily (120). On the contrary, the use of cabergoline, a long-acting ergoline D2 agonist derivative, has been proved to improve ovarian response in hyperprolactinemic patients with PCOS candidates for treatment with gonadaotrophins (121). These data suggested the presence of a dopaminergic component in the control of LH release in PCOS patients (121). 11. Conclusion Ovulation induction in women with PCOS who present with CC-resistant anovulatory infertility remains a major challenge in gynecologic endocrinology. Traditional alternatives for CC-resistant patients include gonadotrophin therapy and laparoscopic ovarian diathermy. However, because of the cost and risk inherent in these therapies, alternative treatments are attractive. Obese PCOS women should try to attain BMI<30kg/m2 prior to commencing ovulation induction therapy. In view of our experience, combined metforminCC therapy did result in modest ovulation and pregnancy rates. Accordingly, it is logical to offer combined metformin- CC therapy for CC-resistant PCOS patients before resorting to more expensive alternatives especially in developing communities where economic aspects of therapy are important. Third generation aromatase inhibitors are promising agents for treatment in these patients. Figure 1 shows an algorithm for ovulation induction treatment in anovulatory infertile women with CC-resistant PCOS. CC- resistant PCOS Obese women Non Obese women • Metformin+ CC (3-6 cycles) Optimize lifestyle • 3rd generation AIs (Letrozole or anastrozole (3-6 cycles) = Weight loss (Diet + exercise) if BMI >29 Kg/m2 LOD Gonadotrophins CC: Clomiphene Citrate AIs: Aromatase inhibitors LOD: laparoscopic ovarian drilling Failure In vitro fertilization Fig. 1. 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A prospective, doubleblind, randomized, placebo-controlled clinical trial of bromocriptin in clomipheneresistant patients with polycystic ovary syndrome and normal prolactin level. Archives of Gynecology and Obstetrics, Vol. 269, No.2 ,pp.125-129. [121] Papaleo, E.; Doldi, N.; De Santis, L.; Marelli, G.; Marsiglio, E.; Rofena, S. & Ferrari, A. (2001). Cabergoline influences ovarian stimulation in hyperprolactinaemic patients with polycystic ovary syndrome. Human Reproduction. Vol. 16, No.11, pp.22632266. www.intechopen.com Polycystic Ovary Syndrome Edited by Dr. Srabani Mukherjee ISBN 978-953-51-0094-2 Hard cover, 168 pages Publisher InTech Published online 24, February, 2012 Published in print edition February, 2012 Brought into the limelight many decades ago, Polycystic Ovary Syndrome (PCOS) is still, to date, surrounded by controversy and mystery. Much attention has been attracted to various topics associated with PCOS research and there has been a healthy advance towards bettering the understanding of the many implications of this complex syndrome. A variety of topics have been dealt with by a panel of authors and compiled in this book. They span methods of diagnosis, reproductive anomalies, metabolic consequences, psychological mindset and ameliorative effects of various lifestyle and medical management options. These books are designed to update all associated professionals on the recent developments in this fast-growing field and to encourage further research into this thought-provoking subject. How to reference In order to correctly reference this scholarly work, feel free to copy and paste the following: Hatem Abu Hashim (2012). Management of Women with Clomifene Citrate Resistant Polycystic Ovary Syndrome – An Evidence Based Approach, Polycystic Ovary Syndrome, Dr. Srabani Mukherjee (Ed.), ISBN: 978-953-51-0094-2, InTech, Available from: http://www.intechopen.com/books/polycystic-ovarysyndrome/management-of-women-with-clomifene-citrate-resistant-polycystic-ovary-syndrome-an-evidencebased-app InTech Europe InTech China University Campus STeP Ri Unit 405, Office Block, Hotel Equatorial Shanghai Slavka Krautzeka 83/A No.65, Yan An Road (West), Shanghai, 200040, China 51000 Rijeka, Croatia Phone: +385 (51) 770 447 Phone: +86-21-62489820 Fax: +385 (51) 686 166 Fax: +86-21-62489821 www.intechopen.com © 2012 The Author(s). Licensee IntechOpen. This is an open access article distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 

W4287811586.txt

ISSN: 2320-5407 Int. J. Adv. Res. 8(04), 678-682 Journal Homepage: - www.journalijar.com Article DOI: 10.21474/IJAR01/10828 DOI URL: http://dx.doi.org/10.21474/IJAR01/10828 RESEARCH ARTICLE PREVALENCE ESTIMATES OF VAGINAL CANDIDIASIS IN NORTHERN AND WESTERN REGIONS, SAUDI ARABIA WITH REFERENCE TO DIABETES MELLITUS RELATION Arwa Alzarani, Renad Almusawi, Shuruq Alshammari and Soha Abdallah …………………………………………………………………………………………………….... Manuscript Info Abstract ……………………. ……………………………………………………………… Manuscript History Received: 14 February 2020 Final Accepted: 16 March 2020 Published: April 2020 Key words:Vaginal Candidiasis, Diabetes Mellitus Vulvovaginitis, Objective: To identify and determine the prevalence, frequency and factors that cause vaginal Candidiasis among women and how that related to diabetes mellitus in some cases. Methods: Cross-sectional- analytic study was conducted from September 2019 till March 2020 at Hail and Almandine regions, KSA, using questionnaire. statistical analysis was carried out by SPSS 22. Results: The study finding showed that (41,3%) from participants were(30-40y), (34,4%) from(40-50y), (18.3%) from (20-30 y) and (5%) from(50-60y). As for the symptoms accompanied by vaginitis and vulvovaginal Candidiasis, the study found that (68%) of the participants suffering from abdominal pain, constipation, diarrhea, bloating. Other signs like vaginal odor (44%) white thick discharge from the vagina were very high (72.8%) of female participants. (45%)44 felt itching and burning sensation and for pain during sex showed (32.9%) and with bleeding (6.6%). As factors that help repeat infection, prolonged courses of antibacterial drugs by (11%) of the responders while the use of vaginal douching increase the incidence of infection by (56.3%) and diabetes mellitus was the major factor which plays a role in incidence and recurrence of infection by (40.4%). Conclusion: Spreading awareness among women about the factors cause recurrence of vaginal Candidiasis can help control and prevention of such conditions, for diabetic patients more measurement should be taken to control recurrence and health care providers should educate women about risk factors. Copy Right, IJAR, 2020,. All rights reserved. …………………………………………………………………………………………………….... Introduction:Vaginitis is one of the major problems facing women. The common cause of the condition is by infection with yeast (Curry et al.1994).Candida Spp., are one of pathogens associated with these condition and Candida albicans is the mostly isolated one. women with diabetes suffering from fungal vaginitis and vulvovaginitis and the intensity of condition is more sever, resistant and recurrent are common (Wilson et al.1987). The aim of this study is to determine the prevalence of Candida vaginitis among women with special reference to diabetic cases in Hail and Al Medina regions, Saudi Arabia from September 2019 till March 2020. Corresponding Author:- Arwa Alzarani 678 ISSN: 2320-5407 Int. J. Adv. Res. 8(04), 678-682 Material and Methods:Study Design: Cross sectional -surveillance study Study population: Women / all age/married or not married/diabetic or not Study strategies: A standardized questionnaire was distributed to all women aged from 18 and above. Study sampling: A total of 334 participated in this study. Data collection: Data was collected by using standardized questionnaire via social media, email and hard copies. Analysis of data: Data was analyzed using Statistical Package for Social Sciences (SPSS) program 22 and Microsoft Excel-97 software. Ethical consideration: Informed Consent was addressed during this study. Results:Regarding participant profile, the study finding showed that (41,3%)from participants were(3040y),(34,4%)from(40-50y),(18.3%) from (20-30 y) and(5%) from(50-60y). (Fig.,1) It observed that most of responders were married (85%), and only (15%) were single. (Table 1). Table 1:- Marital status of responder. Frequency Valid married 284 single 50 Total 334 Percent Valid Percent 85.0 15.0 100.0 85.0 15.0 100.0 Cumulative Percent 85.0 100.0 679 ISSN: 2320-5407 Int. J. Adv. Res. 8(04), 678-682 As for the symptoms accompanied vaginitis and vulvovaginal candidiasis the study found that (68%) of the participants suffering from abdominal pain, constipation, diarrhea, bloating while (32%) have no signs. Other signs like vaginal odor (44%)white thick discharge from vagina where very high (72.8%) of females and (27.2%) with no discharge (56%) no odor. (45%) felt itching and burning sensation while (54%) with no burning sensation. For pain during sex showed (32.9%) and with bleeding (6.6%) (Table 2). Table 2:- Symptoms accompanied vaginitis. 1-Do you suffer with constipation, diarrhea, bloating or abdominal pain Frequency Percent Valid Percent Cumulative Percent Valid yes 227 68.0 68.0 68.0 no 107 32.0 32.0 100.0 Total 334 100.0 100.0 2-Do you notice a white thick discharge from your vagina Frequency Percent Valid Percent Cumulative Percent Valid yes 243 72.8 72.8 72.8 no 91 27.2 27.2 100.0 Total 334 100.0 100.0 3-During the past week, have you been bothered by: Itching or burning sensation in your Vagina Frequency Percent Valid Percent Cumulative Percent Valid yes 151 45.2 45.2 45.2 no 183 54.8 54.8 100.0 Total 334 100.0 100.0 4-Your vulvovaginal symptoms causing pain during sexual activity Frequency Percent Valid Percent Cumulative Percent Valid yes 110 32.9 32.9 32.9 no 224 67.1 67.1 100.0 Total 334 100.0 100.0 5-Your vulvovaginal symptoms causing bleeding during sexual activity Frequency Percent Valid Percent Cumulative Percent Valid yes 22 6.6 6.6 6.6 no 312 93.4 93.4 100.0 Total 334 100.0 100.0 6-Odor from your Vagina Frequency Percent Valid Percent Cumulative Percent Valid yes 147 44.0 44.0 44.0 no 187 56.0 56.0 100.0 Total 334 100.0 100.0 Regarding factors that can cause vaginitis and frequency of infection,the results showed that wear nylon or synthetic pantyhose or underwear which make the vaginal area not dry and not well airyated by (22%) and prolonged courses of antibacterial drugs by (11%) of the responders while the use of vaginal douching increase the incidence of infection by (56.3%). Diabetes one of the major factors which play a role in incidence and recurrence of infection by (40.4%). (Table 3). Table 3:- Factors help in inducing infection and increase frequency . 1-Do you wear nylon or synthetic pantyhose or underwear Frequency Percent Valid Valid Percent Cumulative Percent 680 ISSN: 2320-5407 Int. J. Adv. Res. 8(04), 678-682 yes 74 22.2 22.2 no 260 77.8 77.8 Total 334 100.0 100.0 2-Have you taken repeated or prolonged courses of antibacterial drugs Frequency Percent Valid Percent Valid yes 37 11.1 11.1 no 297 88.9 88.9 Total 334 100.0 100.0 3-Vagina douching Frequency Percent Valid Percent Valid yes 188 56.3 56.3 no 146 43.7 43.7.0 Total 334 100.0 100.0 4-Diabetic patient Frequency Percent Valid Percent Valid yes 135 40.4 40.4 no 199 59.6 59.6 Total 334 100.0 100.0 22.2 100.0 Cumulative Percent 11.1 100.0 Cumulative Percent 56.0 100.0 Cumulative Percent 40.4 100.0 Discussion:Self-hygiene associated infections specially in women are very important issue globally. Vaginal Candidiasis is an irritated infection specially when it recurrently happened, the factors that help the recurrence is varies depend on persons habits such using nylon underwear and daily routine. In agreement with(Moraesetal 1998)as he reported thatPersonal hygiene is an important factor helps in prevent recurrence of infection and successful treatment, also he believes that preventing and controlling of background factors conceders major steps in successful treatment. As in the presence study results showed that vaginal douching plays a role in increasing the incidence of recurrence (Hacialiogluet al and Sen et al 2009)also reported that vaginal douching (VD) as traditional practice changes the PH of the vagina which affect the vaginal mucosal immune response leading to repetitive infection.(Scudamore et al 1992)reported that Diabetes mellitus has long been considered as one of the factors causing Candida vaginitis that also highly agreed with this study results but should be more investigation on the type of diabetes mellitus and how it can affect the recurrence as mentioned by(De Leo et al 2002)that Type 1 and type 2 diabetes specially type 2 can be a risk factor for vaginal Candida colonization in women also (Bohannon et al 1998)stated thatthe basic problem of vulvovaginitis in women with uncontrolled blood glucose levels and the control of blood glucose level with suitable antifungal treatment play important role in controlling vaginal Candida infection on diabetic women.One of the complications experienced by women who have diabetes is Candida vaginitis some believes that condition caused by malfunctioning leucocytes with those with uncontrolled blood glucose levels reported by (Raithet al 1983). Fungal vaginitis is more disturbing in sever hyperglycemic conditions founded by (Chaponiset al 1993. Ozraet al 2007)believes that causes of recurrence were due to false diagnosis of pathogenic organism but on the other hand different from(Forster et al 1998) who mentioned that even when correct diagnosis had been made background factors especially those who have diabetes can cause treatment failure. Other important factors as the presence study finding isAntibiotic use, which is a short-term risk factor for symptomatic vulvovaginal candidiasis, either as a first episode or in the form of recurrence. Increasing duration of antibiotic use is directly related with an increased prevalence of Candida vaginal infection as mentioned by (Spinilloet al1999).In accordance with the findings of the present study, the prevalenceCandida vaginitis high among women who delayed diagnosis and treatment can lead to poor patient outcomes and high medical costs. The overall burden of fungal diseases is challenging to quantify, because they are likely substantially underdiagnosed as agreed with(Benedict et al 2019). Conclusion:In conclusion spread awareness among women about the factors cause recurrence of Candida infection of the vagina, can help control and prevention of such conditions, for diabetic patients more measurement should be taken to control recurrence and health care providers should educate women about risk factors. 681 ISSN: 2320-5407 Int. J. Adv. Res. 8(04), 678-682 Recommendation:Ore investigation has to be done especially the relation between diabetic patients and vaginal Candidiasis to know more about which type of diabetes help more in recurrence, the Candida infection type Albicans and non albicans and frequency of vaginal douching, which help in given the specific treatment to prevent and control recurrence. References:1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Benedict K, Jackson BR, Chiller T, Beer KD. Estimation of Direct Healthcare Costs of Fungal Diseases in the United States. 2019;68(11):1791–7. Candida-albicans-abtotungsaktivitat V. Decreased Candida albicans Killing Activity of Granulocytes from Patients with Diabetes mellitus. 1983;26(April):557–64. Chaponis R, Bresnick P, Weiss R, Edwards L. Candida vaginitis: signs and symptoms aid women's self recognition. J Clin Res Drug Dev 199;7: 17-23. Curry SL, Barclay DL. Benign disorders of the vulva and vagina. In: DeCherney AH, Pernoll ML, eds. Current Diagnosis and Treatment of Obstetrics and Gynecology, 8th Edition. Norwalk, Connecticut: Appleton & Lange, 1994;689–700. de Leon EM, Jacober SJ, Sobel JD, Foxman B. Prevalence and risk factors for vaginal Candida colonization in women with type 1 and type 2 diabetes. BMC Infect Dis. 2002;2:1–6. Foster DW. Diabetes mellitus. In. Fauci AS, Braunwald E, Isselbacher KJ, et al., Eds. Harrison's Principles of Internal Medicine, 14th Edition. New York: McGraw-Hill, 1998;2060–81. Malazy OT, Shariat M, Heshmat R, Majlesi F. V ULVOVAGINAL C ANDIDIASIS AND ITS R ELATED F ACTORS IN D IABETIC W OMEN. 2007;46(4):399–404. Moraes PS. Recurrent vaginal candidiasis and allergic rhinitis: a common association. Ann Allergy Asthma Immunol 1998;81:165–9. Paper C. A descriptive study of douching practices in Turkish women. 2009;57–64. Sakru N, Inceboz T, Inceboz U, Zeren I, Karakus M, Kirca U. Does vaginal douching affect the risk of vaginal infections in pregnant women? Saudi Med J. 2006;27(2):215–8. Scudamore JA, Tooley PJ, Allcorn RJ. The treatment of acute and chronic vaginal candidosis. Br J Clin Pract1992;46:260–3. Spinillo A, Capuzzo E, Acciano S, Santolo A De, Zara F. Effect of antibiotic use on the prevalence of symptomatic vulvovaginal candidiasis. 1999;696:14–7. Susceptibility I, Vulvovaginal TO. Treatment of Vulvovaginal Candidiasis in Patients With Diabetes. 1998;21(3):451–6. Wilson CAB, Hardmant N, Gamblinii SJ. Yeast phosphoglycerate kinase : investigation of catalytic function by site-directed mutagenesis. 1987;241:609–14. 682 

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1 Inhibition of Mitochondrial Carrier Homolog 2 (MTCH2) Suppresses Tumor 2 Invasion and Enhances Sensitivity to Temozolomide in Malignant Glioma 3 4 Authors: Qiuyun Yuan1,#, Wanchun Yang1,#, Shuxin Zhang1, Tengfei Li1, Mingrong Zuo1, 5 Xingwang Zhou1, Junhong Li1, Mao Li1, Xiaoqiang Xia1, Mina Chen1,*, Yanhui Liu1,* 6 7 Affiliation: 8 1 9 University, Chengdu 610041, People’s Republic of China. 10 # Department of Neurosurgery, State Key Laboratory of Biotherapy, West China Hospital, Sichuan These authors contributed equally to this work. 11 12 *Correspondence: Mina Chen (chenmina2010@scu.edu.cn) and Yanhui Liu (liuyh@scu.edu.cn), 13 Department of Neurosurgery, West China Hospital, Sichuan University, 37 Guoxue Avenue, 14 Chengdu, 610041, Sichuan Province, People’s Republic of China. -1- 15 Abstract 16 Background: Malignant glioma exerts a metabolic shift from oxidative phosphorylation 17 (OXPHOs) to aerobic glycolysis, with suppressed mitochondrial functions. This phenomenon 18 offers a proliferation advantage to tumor cells and decrease mitochondria-dependent cell death. 19 However, the underlying mechanism for mitochondrial dysfunction in glioma is not well 20 elucidated. MTCH2 is a mitochondrial outer membrane protein that regulates mitochondrial 21 metabolism and related cell death. This study aims to clarify the role of MTCH2 in glioma. 22 Methods: Bioinformatic analysis from TCGA and CGGA databases were used to investigate the 23 association of MTCH2 with glioma malignancy and clinical significance. The expression of 24 MTCH2 was verified from clinical specimens using real-time PCR and western blots in our 25 cohorts. siRNA-mediated MTCH2 knockdown were used to assess the biological functions of 26 MTCH2 in glioma progression, including cell invasion and temozolomide-induced cell death. 27 Biochemical investigations of mitochondrial and cellular signaling alternations were performed 28 to detect the mechanism by which MTCH2 regulates glioma malignancy. 29 Results: Bioinformatic data from public database and our cohort showed that MTCH2 30 expression was closely associated with glioma malignancy and poor patient survival. Silencing 31 of MTCH2 expression impaired cell migration and enhanced temozolomide sensitivity of human 32 glioma cells. Mechanistically, MTCH2 knockdown increased mitochondrial oxidative damage 33 and decreased pro-survival AKT signaling. 34 Conclusion: Our work identifies the oncogenic role of MTCH2 in gliomas, and establishes the 35 causal relationship between MTCH2 expression and glioma malignancy, which may provide a 36 potential target for future interventions. 37 Key words: MTCH2, glioma, temozolomide, mitochondria, cell migration, cell death. -2- 38 Abbreviations 39 MTCH2: mitochondrial carrier homolog 2; 40 GBM: glioblastoma; 41 TMZ: temozolomide; 42 OXPHOs: oxidative phosphorylations; 43 TCGA: The Cancer Genome Atlas Research Network; 44 CGGA: the Chinese Glioma Genome Atlas; 45 4-HNE: 4-Hydroxy-2-Nonenal; 46 ROS: reactive oxygen species. -3- 47 Background 48 Originating from glial cells, glioma is a common type of malignant and lethal tumor in the brain, 49 which can be divided into astrocytomas, oligodendrogliomas, and glioblastomas (GBM) (1, 2). 50 Glioma displays aggressive growth and diffuse invasion, and evolves rapidly by acquiring new 51 mutations and oncogene expressions for drug resistance (3). Although current standard care of 52 maximal safe surgical resection followed by radiotherapy and concurrent temozolomide (TMZ) 53 chemotherapy provides survival benefit to selected patients (4), glioma still exhibits poor 54 responses to approved therapies and carries an inferior prognosis. Therefore, molecular 55 understandings of the initiation and progression of glioma are urgently needed. 56 Over the last decades, a well-appreciated hallmark of glioma is the altered tumor metabolism 57 (5, 6), a phenomenon known as Warburg effect (7). This effect fulfills the demands for energy 58 and biomass production of rapid proliferation in glioma cells (6). Extensive studies established 59 that mitochondrial dysfunction is a prominent mechanism by which glioma cells exert its 60 metabolic shift from oxidative phosphorylation (OXPHOs) to glycolysis, regardless of oxygen 61 availability. During this metabolic reprogramming, mitochondrial function is glioma is evidently 62 suppressed through mutations of mitochondrial DNA (8), altered metabolic enzyme atlas (9, 10), 63 and imbalanced morphological dynamics (11). All these events offer a proliferation advantage to 64 tumor cells and decrease mitochondria-dependent cell death to chemical drugs (12). Although a 65 number of studies have increased the understanding of mitochondrial dysfunction in glioma cells, 66 the molecular repertoire is still largely unknown. 67 To explore the mechanistic interplay between mitochondrial dysfunction and glioma 68 progression, we focused on mitochondrial carrier homolog 2 (MTCH2), a critical regulator of 69 mitochondrial metabolism and related cell death (13, 14). MTCH2 is a mitochondrial outer -4- 70 membrane protein that interacts with the truncated BH3-interacting domain death agonist (tBID) 71 to regulate cell apoptosis (15-17). Previous studies demonstrate that loss of MTCH2 impairs 72 mitochondrial architecture and functions, including enlarged size (18), reduced motility (19), and 73 elevated oxidative stress (20). MTCH2 expression was associated with several types of tumors. 74 For example, MTCH2 was down-regulated in ErbB2-driven mammary carcinoma (21), and its 75 induction reduced tumorigenicity and leads to growth arrest of breast cancer (22). However, a 76 recent study showed the oncogenic function of MTCH2 in acute myeloid leukemia (AML). 77 Inhibiting MTCH2 decreased tumor growth and induced differentiation of AML cells (23). 78 However, the role of MTCH2 in malignant glioma is not defined. 79 In this study, we reveal the oncogenic function of MTCH2 in malignant glioma. MTCH2 80 expression is closely associated with glioma progression, and its knockdown impairs cell 81 migration and enhances temozolomide sensitivity of glioma cells. Our work establishes the 82 causal relationship between MTCH2 expression and glioma malignancy, and defines MTCH2 as 83 a potential target for future interventions. 84 85 Methods 86 Patient samples 87 Glioma and adjacent brain tissues included in this study were obtained from primary glioma 88 patients undergoing elective craniotomy at West China Hospital between January 2019 and 89 January 2020. All human studies were approved by the Institutional Review Board of West 90 China Hospital of Sichuan University (project identification code: 2018.569). All patients 91 provided written informed consent. The diagnoses of glioma grade were made by three 92 independent pathologists. Clinical samples were used for detection the mRNA and protein levels. -5- 93 Bioinformatic analysis 94 Biomarker data and clinical information of The Cancer Genome Atlas Research Network (TCGA) 95 and the Chinese Glioma Genome Atlas (CGGA) LGG-GBM cohorts were downloaded from 96 TCGA (https://gdc-portal.nci.nih.gov/) and CGGA (http://www.cgga.org.cn) repository. IDH 97 mutation was determined by merging IDH1 codon 132 and IDH2 codon 172 mutations. 98 Chromosome 1p19q co-deletion was determined by 1p32 and 19q13 segment focal CNV value 99 less than -0.3. MGMT promoter methylation level bins were divided using the 10% and 40% 100 percentiles of mean beta value of probes between 131264896 and 131265737 on chromosome 10 101 (GRCh38 genome). For survival analysis, patients were divided into low and high gene 102 expression groups by median expression level. Hazard ratio for expression was tested using log 103 rank test. Gene expression is presented as normalized FPKM. Correlation and survival analysis 104 were conducted in R using grid and survminer packages. 105 106 Cell culture and siRNAs transfection 107 Human glioblastoma cell lines U-87MG, U-251 MG and A172 cell lines were obtained from 108 Type Culture Collection of the Chinese Academy of Sciences (CTCC, Shanghai, China). Glioma 109 cells were maintained at DMEM (Hyclone) with 10% FBS (PAN), and cultured under 5% CO2 at 110 37°C incubator. All cells were validated by Short Tandem Repeat (STR) analysis. For MTCH2 111 knockdown, targeted siRNA were transfected into cultured glioma cells using Lipofectamine 112 RNAiMAX reagent (Invitrogen) in Opti-MEM (Invitrogen) over 72 hours. The siRNA sequences 113 are as follow: siRNA MTCH2#1: sense 5'-GGUAAAGUUUUACAGCAUUTT-3'; antisense 114 5'-AAUGCUGUAAAACUUUACCAT-3'; 115 5'-GAGCCGAGGAAAUAGCUUATT-3'; antisense 5'- UAAGCUAUUUCCUCGGCUCAT-3'. siRNA -6- MTCH2#2: sense 116 Immunofluorescence 117 For immunofluorescence assay to observe MTCH2 cellular localization, A172 cells were 118 cultured on coverslips over 48 hours. For Mito-Tracker staining, A172 cells were incubated with 119 the Mito-Tracker dye (Invitrogen, 200 nM) in DMEM at 37°C incubator for 15 minutes (mins). 120 Then, cells were fixed with 4% paraformaldehyde (PFA, Sigma) solution at room temperature for 121 30 mins. After permeabilization with 0.25% Triton X-100 in PBS, cells were blocked with 10% 122 goat serum and incubated with primary antibodies (anti-MTCH2, Proteintech) overnight at 4°C. 123 Then cells were washed with PBS for three times and incubated with ALEXA FLUOR 488/594 124 secondary antibodies, and mounted slides using ProLong® Gold Antifade Reagent with DAPI 125 (Invitrogen). Images were acquired using Olympus BX63 microscope. 126 127 Cell apoptosis assay 128 To detect cell apoptosis, we conducted flow cytometry and Hoechst staining in glioma cells 129 treated with temozolomide (Selleck Chemicals, 200 μM). For flow cytometry, glioma cells were 130 cultured with 60~80% density in 6-well plate. After cells were transfected with siRNA-MTCH2 131 and subsequent temozolomide treatment for 48 hours, cells were detached by 0.25% trypsin 132 (Invitrogen) and applied with Annexin V Alexa Fluor647/7-AAD kit (Beijing 4A Biotech) for 133 flow cytometry on a Beckman cytoflex. For Hoechst 33258 staining, cells were cultured on 134 coverslips overnight, and treated as above. Then cells were fixed by 4% paraformaldehyde (PFA) 135 solution and incubated with Hoechst 33258 staining kit (Beyotime) as manufacture instructions. 136 Images were acquired using Olympus BX63 microscope, and cell counting was performed by 137 Image Pro Plus software. 138 -7- 139 Scratch wound healing assay 140 To detect the effect of MTCH2 on cell migration of glioma cells, we performed scratch wound 141 healing assay as previous described (24). Briefly, glioma cells were seeded in 6-wells plate 142 overnight, and then siRNA MTCH2 were transfected into cells when cells reached at least 90% 143 density. Cells were wounded with a 200 μL pipette tip and washed 3 times by PBS to remove 144 detached cells. During the migration, glioma cells were cultured in DMEM without FBS. Images 145 were continuously captured in period days and analyzed by ImageJ software. 146 147 Cell lysis solution and western blots 148 Total cells were lysed with 2% SDS solution plus protease and phosphatase inhibitors (Thermo 149 Scientific). Protein concentrations were tested by BCA kit and equivalent proteins were loaded 150 into SDS-PAGE. Following western blots were performed according to standard procedures. The 151 primary antibodies were list as follow: MTCH2 (Proteintech, Cat#16888-1-AP), Beta actin 152 (Boster Biological Technology, Cat#BM0627), PDHE1-A (Abcam, Cat#ab110334), GFAP 153 (Millipore, Cat#G3893), Tom20 (Santa Cruz Biotechnology, Cat#sc-136211), Tom40 (Abcam, 154 Cat#ab185543), MMP-9 (Cell Signaling Technology, Cat#3852), N-cadherin (Cell Signaling 155 Technology, Cat#4061), Vimentin (Cell Signaling Technology, Cat#5741), 4-HNE (Abcam, 156 Cat#ab48506), DRP1 (BD Biosciences, Cat#611112), phospho-DRP1 (Ser616) (Cell Signaling 157 Technology, Cat#3455), BcL2 (Santa Cruz Biotechnology, Cat#sc-7382), Bax (Millipore, 158 Cat#06-499), phospho-AKT (Thr308) (Cell Signaling Technology, Cat#2965), phospho-AKT 159 (Ser473) (Cell Signaling Technology, Cat#4060), AKT (Cell Signaling Technology, Cat#9272), 160 phospho-S6 (Ser240/244) (Cell Signaling Technology, Cat#2215), S6 (Cell Signaling Technology, 161 Cat#2217) and phospho-4EBP1 (Thr37/46) (Cell Signaling Technology, Cat#9459). -8- 162 RNA extraction and real-time PCR 163 Trizol reagent was used to extract total RNA following manufacturer instructions. RNA reversed 164 transcription using PrimeScriptTM RT reagent Kit (Takara), and analyzed by quantitative PCR 165 (qPCR) using SYBR Premix Ex TaqTM II (Takara) in Bio-Rad iQ5 system. Relative gene 166 expression was normalized to Beta actin. qPCR primers were as follows: hMtch2 F: 167 5'-GGTCTTGTTCCTCGCCTTCT-3'; R: 5'-TGGTAGAAACCCCACTGTCC-3. 168 169 Statistical analysis 170 Statistical analysis was conducted in GraphPad Prism (v6.01, GraphPad Software, Inc). Data 171 were expressed as mean ± SEM. Differences between groups were tested for statistical 172 significance using Student’s t test for two-group comparisons, and one-way analysis of variance 173 (ANOVA) followed by the Tukey post hoc tests for multi-group comparisons. A p-value < 0.05 174 (*) was considered statistically significant, and **indicates p < 0.01, and *** p < 0.001. 175 176 Results 177 MTCH2 expression is associated with glioma malignancy. 178 As a first step to investigate the possible contribution of MTCH2 in gliomagenesis, we analyzed 179 MTCH2 expression profiles across multiple types of human tumors. Interrogation of TCGA 180 Pan-Cancer dataset revealed that MTCH2 was highly expressed in several tumors despite 181 different tissue origins, including cancers in the breast (BRCA), cervix (CESC), esophagus 182 (ESCA), lung (LUSC) and brain (GBM) (Fig. 1a). This data suggests that MTCH2 183 overexpression may be a general mechanism employed by solid tumors. Further examinations 184 confirmed that the mRNA and protein levels of MTCH2 were increased in the tumor compared -9- 185 to matched adjacent tissues in our glioma cohort (Fig. 1b-d). Consistent with the fact that 186 MTCH2 expression increased in glioma tissues, Kaplan-Meier survival analysis for multiple 187 human glioma datasets (TCGA and CGGA) showed that patients with lower MTCH2 expression 188 displayed significantly better overall survival than those with higher MTCH2 expression (Fig. 189 1e-f). Therefore, we conclude that MTCH2 is highly expressed in human gliomas and its 190 expression reciprocally correlates with patient survival. 191 To further investigate the correlation of MTCH2 expression with clinical significance in 192 gliomas, we performed an in silico analysis using gene expression and associated clinical data of 193 glioma cohorts from two independent publicly available datasets, TCGA and CGGA. Results 194 from TCGA showed that MTCH2 expression was positively correlated with glioma grade, and 195 high MTCH2 expression tended to be associated with GBM subtype (Fig. 2a-b). IDH mutation, 196 chromosome 1p/19q co-deletion and MGMT promoter methylation are pivotal biomarkers for 197 the guidance of glioma prognostication and treatment (25). We found that MTCH2 expression 198 was exceedingly associated with IDH wildtype and 1p/19q non-codeletion group, which 199 indicates poor glioma prognosis (Fig. 2a and c). Consistently, data from CGGA confirmed that 200 MTCH2 expression was positively associated with tumor grade, IDH wildtype and 1p/19q 201 non-codeletion in Chinese glioma population (Fig. 2d-g), supporting the notion that MTCH2 202 expression is correlated with glioma malignancy. 203 204 Silencing MTCH2 expression impairs cell migration and enhances temozolomide sensitivity of 205 human glioma cells. 206 Based on the finding that MTCH2 expression is closely correlated with glioma malignancy, we 207 asked whether MTCH2 functionally promotes gliomagenesis. We first examined the expression - 10 - 208 pattern of MTCH2 in glioma cell lines, compared with primary cultured brain cells, including 209 neurons and astrocytes from embryonic mouse. Western blot results showed that MTCH2 protein 210 was extremely high in glioma cell lines, including U-87 MG, U-251 MG and A172 cells, in 211 contrast to primary mouse neurons and astrocytes (Fig. 3a). Noted that all the three glioma cells 212 were GFAP positive, whereas neurons were enriched with PDHE1-A, a metabolic gatekeeper in 213 mitochondrial oxidative phosphorylation (26). We next conducted a knockdown strategy using 214 small interfering RNA (siRNA) to reduce MTCH2 expression in A172 human glioblastoma cells. 215 Real-time PCR results showed that the mRNA level of MTCH2 was dramatically decreased by 216 targeted siRNA (Fig. 3b). Western blot results further confirmed that MTCH2 proteins were 217 decreased by >70% in siRNA-MTCH2 groups (Fig. 3c). MTCH2 is a mitochondrial outer 218 membrane protein. We found that immunofluorescence signals of MTCH2 were well 219 co-localized with Mito-Tracker, and these signals were nearly disappeared by MTCH2 220 knockdown (Fig. 3d). 221 We next examined whether MTCH2 knockdown reduced glioma malignancy. A common 222 feature of glioma is the diffused tumor invasion, and thus we evaluated the effect of MTCH2 223 knockdown on the suppression of glioma cell migration. Images of the wound healing assay 224 showed that MTCH2 knockdown dramatically reduced the migration of A172 cells (Fig. 4a-b). 225 Biochemical results also showed that cellular signaling related to cell migration, including 226 MMP-9, N-cadherin and Vimentin, were decreased by MTCH2 silencing (Fig. 4c-d), indicating 227 that MTCH2 is required for the oncogenic invasion of glioma cells. 228 Then, we investigate the effect of MTCH2 on cell survival in glioma cells. Under basal 229 conditions, MTCH2 knockdown slightly affected the survival of A172 cells by Hoechst staining 230 (Fig. 5a (upper panel) and b (left panel)). However, when cells applied with temozolomide - 11 - 231 (TMZ), we found that MTCH2 knockdown dramatically increased cell death (Fig. 5a (lower 232 panel) and b (right panel)). Moreover, data from flow cytometry showed similar results that 233 MTCH2 knockdown cells were more sensitive to TMZ induced death of glioma cells (Fig. 5c-d). 234 All these data suggest that MTCH2 is indispensable to glioma malignancy, and MTCH2 235 silencing impairs cell migration and enhances TMZ sensitivity. 236 237 MTCH2 regulates mitochondrial metabolism and cell survival signaling in glioma cells. 238 MTCH2 is reported to play a critical role in mitochondrial energetic metabolism (20, 27) and 239 associated cell apoptosis involving tBID (15-17). Building on the observation that MTCH2 240 knockdown increases cell apoptosis by TMZ, we tested if MTCH2 regulates mitochondrial 241 related cell survival/death pathways. We first examined the signaling relative to mitochondrial 242 functions. Western blots showed that 4-Hydroxy-2-Nonenal (4-HNE), a marker of lipid 243 peroxidation by reactive oxygen species (ROS) accumulation (28), was significantly increased 244 by MTCH2 knockdown, indicating that loss of MTCH2 induces oxidative stress (Fig. 6a-b). 245 Consistent reduction of DRP1 and its phosphorylation form were found by MTCH2 knockdown, 246 and mitochondrial TCA cycle enzyme PDH was not affected (Fig. 6a-b). These results are 247 supported by previous findings that MTCH2 suppresses mitochondrial fusion (18) and does not 248 influence TCA cycle activity (23). 249 AKT, a serine-threonine kinase, can function to either reduce cellular destruction from 250 oxidants and supports cell survival via BcL-2/Bax pathways (29). For AKT to be fully activated, 251 it is sequentially phosphorylated at Ser473 and Thr308 by mTORC2 and PDK1 activity, 252 respectively (30). We found that MTCH2 silencing decreased AKT phosphorylations at both 253 Thr308 and Ser473 sites, and consistent reduction of pro-survival protein BcL-2 parallely - 12 - 254 induction of pro-apoptotic protein Bax (Fig. 6a and c). We also noted that the downstream 255 mTORC1 signaling was not altered by MTCH2 knockdown, suggesting that MTCH2 256 controlled-mitochondrial functions and cell survival are not dependently on mTORC1 activity 257 (Fig. 6a and d). Taken together, all these results highlight the role of MTCH2 in mitochondrial 258 metabolism and cell survival, implying that MTCH2 is a potential target for glioma intervention. 259 260 Discussion 261 Mitochondrial dysfunction has long been appreciated as a molecular hallmark of malignant 262 gliomas, and the underlying mechanisms have been popular targets for promising therapeutic 263 interventions (26, 31). The present study demonstrates that MTCH2, a critical protein in the 264 mitochondrial outer membrane, regulates glioma malignancy including tumor migration and 265 chemoresistance. Silencing of MTCH2 in glioma cells reduced cell migration and rendered cells 266 more susceptible to apoptosis induced by temozolomide (Fig. 7). 267 Mitochondrion is surrounded by two membranes, and thus a large number of transport proteins 268 are needed to link the biochemical pathways of the cytosol and mitochondrial matrix. This 269 transport system is comprised of mitochondrial translocases of the outer/inner membrane 270 (TOMs/TIMs), mitochondrial pyruvate carriers (MPCs), and mitochondrial carriers etc. (32). 271 MTCH2 is a member of mitochondrial carriers family and regulates cell apoptosis by modulating 272 the activity of mitochondrial permeability transition pore (33). Previous studies examined the 273 role of MTCH2 in several types of tumors, including mammary carcinomas (21, 22) and acute 274 myeloid leukemia (AML) (23). Intriguingly, the role of MTCH2 in these tumors were opposite, 275 anti-oncogenic in breast cancer and oncogenic in AML. Our study demonstrates that MTCH2 is 276 oncogenic in malignant gliomas. We noted that the mutation frequency of MTCH2 is low in - 13 - 277 glioma (less than 4% from cBioPortal, http://www.cbioportal.org/). Therefore, we assume that 278 the altered gene expression (not genomic mutation) of MTCH2 is the major way to regulate 279 glioma malignancy. 280 Glioma has a strong glycolytic phenotype, and a number of the molecular abnormalities that 281 occur in glioma are known to suppress mitochondrial OXPHOs and promote aerobic glycolysis 282 (34, 35). Indeed, glycolytic enzymes have direct anti-apoptotic actions, and decreased 283 mitochondrial function is associated with inhibition of mitochondria dependent apoptosis. 284 MTCH2 is a repressor of mitochondrial metabolism in hematopoietic system, and loss of 285 MTCH2 increases mitochondrial OXPHOs, accompanied with an increase in mitochondrial size, 286 ATP production and consequently ROS accumulation (27). Consistent with this notion, our 287 findings expand current knowledge of MTCH2 in glioma oncogenesis. We showed that MTCH2 288 knockdown increases products of lipid peroxidation, 4-HNE, in glioma cells as well as the 289 susceptibility to TMZ-induced cell death. Moreover, MTCH2 knockdown decreased the protein 290 level of DRP1, an executor of mitochondrial fission, and led to mitochondrial fusion (18), a 291 morphology that is conducive to OXPHOs (36). Based on these results, we propose that MTCH2 292 knockdown may activate OXPHOs in glioma cells, and render cells more susceptible 293 TMZ-induced apoptosis. 294 295 Conclusion 296 In summary, our work identifies the oncogenic role of MTCH2 in malignant gliomas. Glioma 297 cells with reduced MTCH2 expression have lowered invasion property and are sensitive to 298 temozolomide. Our results supplement current understanding in the mitochondrial biology of 299 gliomas, and provide a potential target for future interventions. - 14 - 300 Declarations 301 Ethics approval and consent to participate 302 All human studies were approved by the Institutional Review Board of West China Hospital of 303 Sichuan University (project identification code: 2018.569). All patients provided written 304 informed consent. 305 306 Consent for publication 307 NA 308 309 Availability of data and materials 310 All relevant original data are available from the corresponding authors, upon reasonable request. 311 312 Competing interests 313 The authors declare no competing interests. 314 315 Funding 316 This work was supported by the grant from Sichuan Province Science and Technology Support 317 Program (2017SZ0006 to Yanhui Liu), and the National Natural Science Foundation of China 318 (31501155 to Wanchun Yang and 81571195 to Mina Chen). 319 320 Authors' contributions 321 Qiuyun Yuan, Wanchun Yang, Mina Chen and Yanhui Liu designed the study and wrote the 322 manuscript; Qiuyun Yuan, Wanchun Yang, Tengfei Li, Mingrong Zuo, Xingwang Zhou, 323 Junhong Li, Mao Li and Xiaoqiang Xia performed the experiments and analyzed the data; 324 Shunxin Zhang conducted the bioinformatic analysis. 325 326 Acknowledgements 327 NA - 15 - 328 References 329 1. 330 6;310(17):1842-50. 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Zaltsman Y, Shachnai L, Yivgi-Ohana N, Schwarz M, Maryanovich M, Houtkooper RH, et al. 374 MTCH2/MIMP is a major facilitator of tBID recruitment to mitochondria. Nature cell biology. 2010 375 Jun;12(6):553-62. PubMed PMID: 20436477. Pubmed Central PMCID: 4070879. 376 18. Bahat A, Goldman A, Zaltsman Y, Khan DH, Halperin C, Amzallag E, et al. MTCH2-mediated 377 mitochondrial fusion drives exit from naive pluripotency in embryonic stem cells. Nature communications. 378 2018 Dec 3;9(1):5132. PubMed PMID: 30510213. Pubmed Central PMCID: 6277412. 379 19. Ruggiero A, Aloni E, Korkotian E, Zaltsman Y, Oni-Biton E, Kuperman Y, et al. Loss of forebrain 380 MTCH2 decreases mitochondria motility and calcium handling and impairs hippocampal-dependent cognitive 381 functions. Scientific reports. 2017 Mar 9;7:44401. PubMed PMID: 28276496. Pubmed Central PMCID: 382 5343590. 383 20. Buzaglo-Azriel L, Kuperman Y, Tsoory M, Zaltsman Y, Shachnai L, Zaidman SL, et al. Loss of Muscle 384 MTCH2 Increases Whole-Body Energy Utilization and Protects from Diet-Induced Obesity. Cell reports. 2017 385 Jan 31;18(5):1335-6. PubMed PMID: 28147285. 386 21. Arigoni M, Barutello G, Riccardo F, Ercole E, Cantarella D, Orso F, et al. miR-135b coordinates 387 progression of ErbB2-driven mammary carcinomas through suppression of MID1 and MTCH2. The American 388 journal of pathology. 2013 Jun;182(6):2058-70. PubMed PMID: 23623609. 389 22. Leibowitz-Amit R, Tsarfaty G, Abargil Y, Yerushalmi GM, Horev J, Tsarfaty I. Mimp, a mitochondrial 390 carrier homologue, inhibits Met-HGF/SF-induced scattering and tumorigenicity by altering Met-HGF/SF 391 signaling pathways. Cancer research. 2006 Sep 1;66(17):8687-97. PubMed PMID: 16951184. 392 23. Khan DH, Mullokandov M, Wu Y, Voisin V, Gronda M, Hurren R, et al. Mitochondrial carrier homolog 2 393 is necessary for AML survival. Blood. 2020 Jul 2;136(1):81-92. PubMed PMID: 32299104. 394 24. 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An MTCH2 404 pathway repressing mitochondria metabolism regulates haematopoietic stem cell fate. Nature communications. 405 2015 Jul 29;6:7901. PubMed PMID: 26219591. 406 28. Esterbauer H, Zollner H, Lang J. Metabolism of the lipid peroxidation product 4-hydroxynonenal by 407 isolated hepatocytes and by liver cytosolic fractions. The Biochemical journal. 1985 Jun 1;228(2):363-73. 408 PubMed PMID: 3160340. Pubmed Central PMCID: 1144994. 409 29. Matsuzaki H, Tamatani M, Mitsuda N, Namikawa K, Kiyama H, Miyake S, et al. Activation of Akt kinase 410 inhibits apoptosis and changes in Bcl-2 and Bax expression induced by nitric oxide in primary hippocampal 411 neurons. Journal of neurochemistry. 1999 Nov;73(5):2037-46. PubMed PMID: 10537063. 412 30. Vadlakonda L, Dash A, Pasupuleti M, Anil Kumar K, Reddanna P. The Paradox of Akt-mTOR 413 Interactions. Frontiers in oncology. 2013;3:165. PubMed PMID: 23802099. Pubmed Central PMCID: 414 3687210. 415 31. Martinez-Outschoorn UE, Peiris-Pages M, Pestell RG, Sotgia F, Lisanti MP. Cancer metabolism: a 416 therapeutic perspective. Nature reviews Clinical oncology. 2017 Feb;14(2):113. PubMed PMID: 28094266. 417 32. Lytovchenko O, Kunji ERS. Expression and putative role of mitochondrial transport proteins in cancer. 418 Biochimica et biophysica acta Bioenergetics. 2017 Aug;1858(8):641-54. PubMed PMID: 28342810. 419 33. Alcala S, Klee M, Fernandez J, Fleischer A, Pimentel-Muinos FX. A high-throughput screening for 420 mammalian cell death effectors identifies the mitochondrial phosphate carrier as a regulator of cytochrome c 421 release. Oncogene. 2008 Jan 3;27(1):44-54. PubMed PMID: 17621274. 422 34. Strickland M, Stoll EA. Metabolic Reprogramming in Glioma. Frontiers in cell and developmental 423 biology. 2017;5:43. PubMed PMID: 28491867. Pubmed Central PMCID: 5405080. 424 35. Lokody I. Metabolism: reprogramming metabolic flux in glioma. Nature reviews Cancer. 2014 425 Nov;14(11):706-7. PubMed PMID: 25291294. 426 36. Yao CH, Wang R, Wang Y, Kung CP, Weber JD, Patti GJ. Mitochondrial fusion supports increased 427 oxidative phosphorylation during cell proliferation. eLife. 2019 Jan 29;8. PubMed PMID: 30694178. Pubmed 428 Central PMCID: 6351101. 429 - 18 - 430 Figures and legends 431 Fig. 1 MTCH2 expression is increased in glioma tissues and indicates poor prognosis. 432 433 (a) A Pan-cancer diagram showing the increased MTCH2 expression in brain gliomas. (b) Real-time PCR 434 results showing increased mRNA level of MTCH2 in glioma tissues compared to paired adjacent brain tissues 435 (n=32). (c-d) Western blots (c) and quantifications (d) showing increased proteins of MTCH2 in glioma tissues 436 compared to paired adjacent brain tissues (n=23). (e-f) Survival analysis using clinical information from TCGA 437 (e) and CGGA (f) dataset. Patients are divided into low and high MTCH2 groups by median expression level. - 19 - 438 Fig. 2 MTCH2 expression is associated with glioma malignancy. 439 440 (a) Correlation between MTCH2 expression (FPKM) and glioma histology and prognostic biomarkers in The 441 Cancer Genome Atlas (TCGA) dataset. Patients (N=681) are sort by MTCH2 expression. Glioma histology 442 and biomarkers of patients are shown in color code. (b) Diagrams showing MTCH2 expressions in glioma 443 histology groups. (c) Diagrams showing MTCH2 expression of gliomas with IDH status in the TCGA dataset. 444 (d-g) Diagrams showing MTCH2 expression of gliomas with tumor grade, histology, IDH and 1p19q status in 445 the CGGA dataset. - 20 - 446 Fig. 3 Characterization of MTCH2 knockdown in glioma cells. 447 448 (a) Western blots showing the enriched expression of MTCH2 in glioma cells compared to primary neurons 449 and astrocytes. (b) qPCR results showing the decreased mRNA level of MTCH2 in A172 cells by 450 siRNA-mediated knockdown (N=3). (c) Western blots showing the decreased protein level of MTCH2 in A172 451 cells by siRNA-mediated knockdown. Noted that other mitochondrial outer membrane proteins Tom20 and 452 Tom40 were not affected by MTCH2 knockdown. (d) Immunostaining images showing the mitochondrial 453 localization of MTCH2 in A172 cells. Noted that MTCH2 signals were dramatically decreased by 454 siRNA-mediated knockdown. 455 - 21 - 456 Fig. 4 MTCH2 knockdown impairs the invasion of glioma cells. 457 458 (a-b) Representative images of wound healing assays showing that MTCH2 knockdown inhibits the migration 459 of A172 cells (N=4). The migration of cells into the wound was assessed at 0, 24, 48 and 72 hours. Scale bar, 460 200 μm. (c-d) Western blots (c) and quantifications (d) showing the decreased protein levels of MMP-9, 461 N-cadherin, Vimentin and MTCH2 in A172 cells after wound healing (N=3). 462 - 22 - 463 Fig. 5 MTCH2 knockdown increases temozolomide-induced apoptosis of glioma cells. 464 465 (a-b) Images (a) and quantifications (b) of Hoechst staining showing that MTCH2 knockdown promotes cell 466 apoptosis of A172 cells after temozolomide treatment (200 μM, 48 hours) (N=3). Scale bar, 20 μm. (c-d) 467 Images (c) and quantifications (d) of flow cytometry showing that MTCH2 knockdown promotes apoptosis of 468 A172 cells after temozolomide treatment (200 μM, 48 hours) (N=3). - 23 - 469 Fig. 6 Molecular alternations of MTCH2 knockdown on mitochondrial and cell survival 470 signaling in glioma cells. 471 472 (a-d) Western blots (a) and quantifications (b-d) showing that MTCH2 knockdown alters proteins of 473 mitochondrial metabolism and cell survival signaling, especially 4-HNE, DRP1, BcL2, Bax and pAKT (N=4). 474 - 24 - 475 Fig. 7 Model. 476 477 A schematic demonstrates that MTCH2 regulates cell invasion/apoptosis of malignant gliomas. - 25 - 

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nature communications Article https://doi.org/10.1038/s41467-022-32022-8 Distinctive roles of translesion polymerases DinB1 and DnaE2 in diversification of the mycobacterial genome through substitution and frameshift mutagenesis Received: 17 March 2022 1234567890():,; 1234567890():,; Accepted: 12 July 2022 Check for updates Pierre Dupuy 1, Shreya Ghosh 2, Oyindamola Adefisayo1,3, John Buglino1, Stewart Shuman2 & Michael S. Glickman 1,3 Antibiotic resistance of Mycobacterium tuberculosis is exclusively a consequence of chromosomal mutations. Translesion synthesis (TLS) is a widely conserved mechanism of DNA damage tolerance and mutagenesis, executed by translesion polymerases such as DinBs. In mycobacteria, DnaE2 is the only known agent of TLS and the role of DinB polymerases is unknown. Here we demonstrate that, when overexpressed, DinB1 promotes missense mutations conferring resistance to rifampicin, with a mutational signature distinct from that of DnaE2, and abets insertion and deletion frameshift mutagenesis in homo-oligonucleotide runs. DinB1 is the primary mediator of spontaneous −1 frameshift mutations in homo-oligonucleotide runs whereas DnaE2 and DinBs are redundant in DNA damage-induced −1 frameshift mutagenesis. These results highlight DinB1 and DnaE2 as drivers of mycobacterial genome diversification with relevance to antimicrobial resistance and host adaptation. Genomic integrity is constantly threatened by DNA damage arising from endogenous cell metabolism and exogenous environmental factors. DNA lesions, when not rectified by dedicated repair systems, can persist, block DNA replication, and induce lethal fork collapse. “Translesion DNA Synthesis” (TLS) is an ubiquitous tolerance pathway by which the blocked replicative polymerase is transiently replaced by an alternative DNA polymerase that traverses the lesion1. In E. coli, DinB (Pol IV) and UmuDC (Pol V) are critical mediators of TLS2,3. In vitro, DinB and UmuDC share common biochemical characteristics that facilitate their in vivo function, including low fidelity, low processivity, lack of proofreading activity, and ability to bypass a variety of lesions4–6. In E. coli, the expression of dinB and umuDC is inducible by DNA damage through the SOS response pathway7 and they respectively confer tolerance to alkylating agents and UV8–10. Because of the flexibility of their active site required for lesion bypass11, TLS polymerases catalyze mutagenesis and play a key role in evolutionary fitness12 or antibiotic resistance in bacteria13 and carcinogenesis in eukaryotes14. In E. coli, DinB and UmuDC are mutagenic, inducing substitution mutations as well as indels15–20. Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), which kills 1.5 million people annually21. The major challenges impeding TB eradication efforts include the lack of short regimens of therapy, likely due to antibiotic tolerance mechanisms, and mutational antibiotic resistance22, which is a substantial global health problem21. Mtb acquires antimicrobial resistance exclusively through chromosomal mutations, in contrast to the widespread mechanism of lateral gene transfer in other pathogens23. Human macrophages, the natural habitat of Mtb, expose the bacterium to diverse stresses, many of which directly damage DNA24–28. Mtb DNA repair pathways, in particular translesion polymerases, represent a promising and underexplored target for new TB drugs due to their role in survival within the host and in antimicrobial resistance13. However, 1 Immunology Program, Sloan Kettering Institute, New York, NY 10065, USA. 2Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA. Immunology and Microbial Pathogenesis Graduate Program, Weill Cornell Graduate School, 1300 York Avenue, New York, NY 10065, USA. e-mail: glickmam@mskcc.org 3 Nature Communications | (2022)13:4493 1 Article the molecular pathways controlling chromosomal mutagenesis in mycobacteria are only partially understood. The replication fidelity of the Mtb chromosome is preserved by the proofreading function of the replicative polymerase DnaE1 that, when mutated, drastically increases mutation frequency29. Mycobacteria do not encode UmuDC but rather another TLS polymerase, a paralogue of DnaE1 called DnaE230,31. In Mtb, dnaE2 expression is dependent on DNA damage response13,32. DnaE2 is involved in UV tolerance as well as UV-induced mutagenesis and also contributes to bacterial pathogenicity and the emergence of drug resistance13,33. To date, DnaE2 is the only non-replicative polymerase known to contribute to chromosomal mutagenesis in mycobacteria. Mycobacterial genomes encode several DinBs paralogs: two in Mtb (dinB1/dinX/Rv1537 and dinB2/dinP/Rv3056) and three in the nonpathogenic model Mycobacterium smegmatis (dinB1/MSMEG_3172, dinB2/MSMEG_2294/MSMEG_1014 and dinB3/MSMEG_6443)30,34. Whereas Mtb dinB2 expression is enhanced by novobiocin35, Mtb dinB1 is part of the SigH regulon36 and its expression is induced by rifampicin35 and during human pulmonary TB infection37. In silico and experimental evidence indicates that DinB1, but not DinB2 nor DinB3, has a C-terminal β clamp binding motif and interacts directly with the β clamp in a heterologous organism38. M. smegmatis DinB1, DinB2, and DinB3 catalyze DNA-templated primer extension in vitro39,40. Initial characterization of a dinB1dinB2 double mutant of Mtb, as well as the expression of the proteins in M. smegmatis, failed to identify a role in DNA damage tolerance, mutagenesis or pathogenicity in vivo38. Here we genetically investigate the contribution of mycobacterial TLS polymerases to DNA damage tolerance, antibiotic resistance, and mutagenesis. We show that DinB1 is highly mutagenic in vivo with a strong ability to incorporate substitution mutations conferring the resistance to rifampicin, one of the main drugs used to treat TB, and with a distinct mutagenic signature compared to DnaE2-catalyzed resistance mutations. We also demonstrate a previously unappreciated role for mycobacterial translesion polymerases in frameshift (FS) mutagenesis, with DinB1 and DnaE2 acting as the primary agents of spontaneous and UV-induced homo-oligonucleotide −1 FS mutagenesis in the mycobacterial chromosome. Results DinB1Mtb activity requires five N-terminal amino acids omitted from the annotated ORF To investigate the role of DinB1 in mycobacteria, we expressed M. smegmatis dinB1 (dinB1Msm) from an Anhydrotetracycline (ATc) inducible promoter (tet promoter). Addition of ATc enhanced the level of dinB1Msm mRNA in cells by 1000-fold (Supplementary Fig. 1a). We found that the expression of dinB1Msm in M. smegmatis caused a substantial growth defect and loss of viability (Fig. 1a, b) that was proportional to the level of inducer (Supplementary Fig. 1b, c). dinB1Msm expression also caused DNA damage, as evinced by an increase in the steady-state level of the RecA protein at 4 h post-induction by ATc (Fig. 1c). The effects of expression of M. smegmatis dinB1 contrast with the lack of similar findings when the Mtb gene was expressed38, despite an overall identity of the two proteins of 75% (Supplementary Fig. 2). We found that expression of the Mtb dinB1 gene (dinB1Mtb) in M. smegmatis did not phenocopy dinB1Msm (Fig. 1d). However, we reanalyzed the annotation of the dinB1 open reading frames from M. smegmatis and Mtb and found an alternative translational start codon fifteen nucleotides upstream of the annotated start codon of Mtb dinB1 used in prior experiments (Fig. 1e and Supplementary Fig. 2). Expression of the mRNA encoding this longer form of the Mtb DinB1 (dinB1Mtb+5aa) was induced 250-fold by ATc addition (Supplementary Fig. 1a) and impaired M. smegmatis growth (Fig. 1d), suggesting that the first five amino acids of DinB1 are essential for in vivo activity. These results indicate that prior conclusions about lack of in vivo activity of Mtb DinB1 are attributable to expression of a truncated protein. Nature Communications | (2022)13:4493 https://doi.org/10.1038/s41467-022-32022-8 A catalytic dead mutant of dinB1Msm (dinB1D113A), which is unable to catalyze DNA synthesis in vitro40, exacerbated growth and viability defects compared to the WT polymerase (Fig. 1a, f). In contrast, expression of a dinB1Msm mutant lacking its β clamp binding domain (dinB1Δβclamp), predicted to not interact with the replicative machinery, did not cause growth inhibition or cell death (Fig. 1f, g). These results suggest that DinB1 interacts with the replicative machinery in vivo and competes with the replicative DNA polymerase at replication forks, as proposed in E. coli41,42. We cannot exclude the possibility that DinB1ΔB-clamp derivative is not as stable as WT and that the DinB1-dependent growth defect is unrelated to replication. DinB1 is an error-prone polymerase inducing antibiotic resistance through a characteristic mutagenic signature Because of their active site flexibility required for lesion bypass, most translesion polymerases are error prone11. To determine the mutagenic capability of mycobacterial DinB1 as well as its ability to induce antibiotic resistance, we measured the frequency of rifampicin resistance (rifR), conferred by substitution mutations in the rpoB gene, in strains with temporally controlled expression of dinB1Msm, dinB1Mtb, or dinB1Mtb+5aa. In the strains carrying the empty vector or the dinB1Mtb plasmid, we respectively detected an average of 5.5 and 3.1 rifR/108 CFU 16 h after inducer addition (Fig. 2a). The expression of dinB1Msm or dinB1Mtb+5aa increased the frequency of rifR by six- or eightfold but the expression of dinB1Δβclamp had no effect, suggesting that an interaction between DinB1 and the replicative machinery is required for DinB1-dependent mutagenesis. We observed a similar induction of mutagenesis after dinB1Msm expression in ΔrecA and ΔdnaE2 backgrounds (Supplementary Fig. 1d), showing that the effect of dinB1 on mutation frequency is not the consequence of the RecA-dependent DNA damage response or the previously defined role of DnaE2 in mutagenesis13, further strengthening the conclusion that DinB1 can be directly mutagenic. To determine the mutation spectrum induced by DinB1, we sequenced the rifampicin resistance determining region (RRDR) of the rpoB gene (Fig. 2b). In absence of dinB1 expression, the majority of RRDR mutations were either G>A or C>T (37%) or A>G or T>C (28%), with a minority of other mutations. Expression of dinB1Msm and dinB1Mtb+5aa strongly enhanced the relative proportion of A>G or T>C. The absolute frequency of these mutations was increased by 18- and 21fold after dinB1Msm and dinB1Mtb+5aa expression, respectively. Around 75% of rpoB mutations found after dinB1 expression were in the second nucleotide of the His442 codon compared with 25% in control cells (Fig. 2c and Supplementary Table 4). The mutation was almost exclusively CAC>CGC (His>Arg) and its absolute frequency was increased 21- and 28-fold after dinB1Msm and dinB1 Mtb+5aa expression, respectively (Fig. 2d and Supplementary Table 4). These results demonstrate that DinB1 is prone to induce mutations in vivo, with a characteristic mutagenic signature, A>G or T>C transition mutations, that contributes to rifampicin resistance at a specific codon in RpoB. DnaE2 but not DinBs mediates stress-induced substitution mutagenesis The intrinsic mutagenicity of DinB1 demonstrated above supports a role for the enzyme in chromosomal mutagenesis at high copy number. We next measured the relative contributions of mycobacterial TLS polymerases (DinBs and DnaE2) in spontaneous mutagenesis by characterizing M. smegmatis cells lacking dnaE2, all dinBs, or all known translesion polymerases (dnaE2 + dinBs). In the WT strain, we detected around 5 rifR/108 CFU (Fig. 3a) and 30% of rpoB mutations found in rifR colonies were A>G or T>C, 27% were G>A or C>T, and the remainder distributed across other mutation types (Fig. 3b). The deletion of TLS polymerases did not alter the frequency of rifR or shift the proportion of mutation types found in the 2 Article https://doi.org/10.1038/s41467-022-32022-8 b 0.01 *** ** *** ** ** 0.001 0 d 20 30 Time (hour) 40 ** 0.1 *** *** ** 0.001 0 e 10 20 30 Time (hour) 40 *** Empty tet-dinB1Msm tet-dinB1Msm RpoB 105 Empty tet-dinB1Mtb tet-dinB1Mtb+5aa 0.01 Empty RecA 106 50 0 f 1 OD600 10 * 24h RpoB 50 108 107 4 8 Time (hour) 16 12 g 1 *** *** OD600 *** *** p=0.06 107 Empty tet-dinB1Msm tet-dinB1Δβclamp 0.1 106 0.01 105 0.001 p 0.1 *** 4h RecA am *** 0h 108 cl OD600 1 c 109 Δβ 10 Viability (CFU/ODU) Empty tet-dinB1Msm tet-dinB1D113A te Em t-d p te inB ty t-d 1 M te inB sm t-d 1 D 1 in B 1 13A 100 Viability (CFU/ODU) a *** *** 0 5 10 15 Time (hour) * ** *** *** *** *** 20 25 GGT ATG GAG GGC ACC GTC GCT CGG ACC GCG AGC CGC AGA TGG GTC CTG CAT CTG GAC ATG GAC >>> ..G ..M ..E ..G ..T ..V ..A ..R ..T ..A ..S ..R ..R ..W ..V ..L ..H ..L ..D ..M ..D >>> dinB1Msm TCG GTA GCC CCA AAT AGC ATC ACG GGT GTG GAG TCC CGC TGG GTG CTG CAC CTG GAC ATG GAT >>> ..S ..V ..A ..P ..N ..S ..I ..T ..G ..V ..E ..S ..R ..W ..V ..L ..H ..L ..D ..M ..D >>> dinB1Mtb Fig. 1 | DinB1Mtb activity requires five N-terminal amino acids omitted from the annotated ORF. a, d, g Growth and b, f viability of strains carrying an inducible (tet = Anhydrotetracycline inducible promoter) DinB1 or its indicated derivatives (Msm = M. smegmatis, Mtb = annotated M. tuberculosis DinB1, Mtb+5aa = N terminal extended DinB1, dinB1 D113A = catalytically inactive M. smegmatis DinB1, Δβ clamp (ΔQESLF: 356–360 amino acids of M. smegmatis DinB1)) in presence of inducer. For a, the plotted OD value is the result of continuous dilution to maintain log phase growth (see methods). The viability in f was measured 24 h after inducer addition. c Anti-RecA/RpoB immunoblot from indicated strains with indicated times of inducer treatment. e Alignment of Msm and Mtb DinB1 N-termini with the potential start codons underlined. The blue boxed valine corresponds to the start codon of the published DinB1 noted as DinB1Mtb above, whereas the red boxed valine shows an alternative start codon of an extended DinB1 denoted as DinB1Mtb+5aa. Results shown are means (±SEM) of biological triplicates (a, b, d, g) or from biological replicates symbolized by gray dots (f). Stars above or under the means mark a statistical difference with the reference strain (empty vector) and lines connecting two strains show a statistical difference between them (*, P < 0.05; **, P < 0.01; ***, P < 0.001). p values were obtained on log-transformed data by one-way (f) or twoway (a, b, d, and g) ANOVA with a Bonferroni post-test. rpoB gene (Fig. 3a, b), indicating that the activities of DnaE2 and DinBs are not the predominant mediators of substitution mutagenesis in the basal conditions tested. By analyzing our recently published transcriptomic data32, we found that the expression of dinB1, dinB3, and dnaE2 was induced by UV and ciprofloxacin (cip) in M. smegmatis whereas dinB2 expression was unaffected (Supplementary Fig. 3). The expression level of dinB1 and dinB2 in UV-irradiated cells was not impacted by recA deletion (Supplementary Fig. 3). By contrast, UV induction of dinB3 and dnaE2 expression was reduced in the ΔrecA mutant, a result we confirmed by RT-qPCR (Supplementary Fig. 3). We next investigated the relative contribution of DinBs and DnaE2 to stress-induced mutagenesis by measuring the frequency of rifR in strains exposed to UV, hydrogen peroxide (H2O2) and methyl methanesulfonate (MMS). In the WT strain, we found 108-, 16-, and 24-fold increases of the rifR frequency after treatment with UV, H2O2, and MMS, respectively (Fig. 3a). All three mutagens increased the relative and absolute frequencies of G>A or C>T mutations in the RRDR whereas UV also enhanced A>C or T>G mutations and H2O2 increased G>C or C>G mutations (Fig. 3b). Induction of the rifR frequency by UV and H2O2 was DnaE2-dependent, declining by 10- and 4-fold in ΔdnaE2 cells compared to WT, whereas MMS-induced mutagenesis was not impacted (Fig. 3a). All types of UV- and H2O2-induced rifR mutations (G>A or C>T, A>C or T>G and G>C or C>G) were reduced by the dnaE2 deletion (Fig. 3b). In contrast, the dinB123 deletion did not significantly decrease the mutation frequency in cells treated with UV, H2O2, and MMS or change the spectrum of mutation types (Fig. 3a, b). The analysis of rpoB mutations incorporated by DnaE2 during oxidative stress (WT vs ΔdnaE2) revealed that, unlike DinB1, DnaE2 conferred rifampicin resistance by mutating diverse rpoB codons (Fig. 3c and Supplementary Table 4). Particularly, we found that the presence of DnaE2 increased the absolute mutation frequency in Ser447(TCG>TTG), Ser438(TCG>TTG), His442(CAC>TAC), His442(CAC>GAC), and Asn435(AAC>AAG), but not the DinB1 associated mutation His442(CAC>CGC). These results show that DnaE2, but not DinBs, contributes to UVand H2O2-induced substitution mutagenesis with a distinct mutation spectrum from the DinB1 signature. Nature Communications | (2022)13:4493 Redundancy of DinB1 and DnaE2 in tolerance to alkylation damage and N2-dG adducts The mutagenic properties of DinBs are intimately linked to their active site flexibility required in lesion bypass, a property that 3 Article b p=0.010 *** *** RifR/108 CFU 10 1 Em Empty: 2.2 1.1 tet-dinB1Msm: 0.0 2.2 tet-dinB1Mtb+5aa: 4.2 0.0 n=45 tetdinB1Msm n=24 tetdinB1Mtb+5aa 15 a te p t te -din ty t-d B i n 1 Ms m B1 M a 5a tb + te t p te -din ty t-d B in 1 Msm B te 1 Δβcl t-d am te p t-d inB in 1 Mt b B1 M Em c Empty 30 0 0.1 n=92 5a RifR/108 CFU 100 45 tb + a https://doi.org/10.1038/s41467-022-32022-8 A>T or T>A A>G or T>C A>C or T>G G>A or C>T 2.2 4.3 0.0 4.4 0.0 0.0 G>T or C>A G>C or C>G del. no rpoB mut. 16.3 2.2 4.2 AGC CAG CTG TCG CAG TTC ATG GAC CAG AAC AAC CCG CTG TCG GGT 425 ..S ..Q ..L ..S ..Q ..F ..M ..D ..Q ..N ..N ..P ..L ..S ..G 25.0 25.0 6.7 77.8 4.2 75.0 0.0 2.2 0.0 4.3 0.0 4.2 4.3 1.1 2.2 0.0 0.0 0.0 CTG ACC CAC AAG CGT CGT CTT TCG GCG CTG GGC CCC ....... ACC 440 ..L ..T ..H ..K ..R ..R ..L ..S ..A ..L ..G ..P (+28aa) ..T 1.1 0.0 0.0 1.1 0.0 4.2 CCT GAG GGT CCC AAC ATC GGT CTG ATC GGT TCG 480 ..P ..E ..G ..P ..N ..I ..G ..L ..I ..G ..S rpoB mut./108 CFU d 45 1.1 0.0 0.0 10.9 2.2 4.2 del. no rpoB mut. His442(CAC>CGC) other min. mut. 30 15 We next investigated the role of DnaE2 and DinBs in the tolerance to alkylation damage, as reported for E. coli DinB8,10, by testing the sensitivity of M. smegmatis TLS polymerase mutants to the chemical methylating agents MMS and methylnitronitrosoguanidine (MNNG). Using disc diffusion assays, we found that the ΔdinB123 or ΔdnaE2 mutants were not more sensitive than the WT strain to MMS but the loss of the four TLS polymerases in combination conferred higher sensitivity (Fig. 4a), indicating substantial redundancy. We obtained similar results by plating serial dilutions of these strains on agar medium containing MMS (Supplementary Fig. 4f) and with disc diffusion using MNNG (Fig. 4c). The ΔdnaE2dinB123 MMS and MNNG sensitivity was partially complemented by the introduction of an ectopic copy of dnaE2Msm or dinB1Msm but not dinB2Msm or dinB3Msm (Fig. 4b, d and Supplementary Fig. 4f, g). The ΔdnaE2dinB123 sensitivity to MMS and MNNG was partially complemented by an ectopic copy of dinB1Mtb but not dinB2Mtb (Fig. 4b, d), indicating conservation of DinB1 activities between fast- and slow-growing mycobacteria. Finally, we measured the effect of loss of dinBs and dnaE2 on tolerance to N2-dG adducts. None of the translesion polymerase mutants was impacted in their tolerance to 4-nitroquinoline -1oxide (4NQO) (Supplementary Fig. 4e). Whereas the ΔdinB1, ΔdinB2, and ΔdnaE2 single mutants were slightly more sensitive than the WT strain to nitrofurazone (NFZ)10, the ΔdinB123 and ΔdnaE2dinB123 mutants were highly sensitive (Fig. 4e). Ectopic expression of dinB1Msm, dinB3Msm, dnaE2Msm, or dinB1Mtb in the ΔdnaE2dinB123 strain partially reversed the NFZ sensitivity (Fig. 4f), reinforcing the substantial redundancy of translesion polymerases in mycobacteria for bypassing damage. These results reveal previously unrecognized roles for DnaE2 and DinB1 in the tolerance to genomic alkylation damage and N2-dG adducts in mycobacteria and suggest a dominant role of DinB1 over the other mycobacterial DinBs in TLS. DinB1 mediates −1 frameshift mutations aa +5 tb te Em t te -din pty t-d B in 1 Ms B1 m M 0 Fig. 2 | DinB1 is an error-prone polymerase inducing antibiotic resistance through a characteristic mutagenic signature. a Rifampicin resistance (rifR) frequency in indicated strains in presence of inducer. Results shown are means (±SEM) of data obtained from biological replicates symbolized by gray dots. Stars above bars mark a statistical difference with the reference strain (empty vector) and lines connecting two strains show a statistical difference between them (***, P < 0.001). p-values were obtained on log-transformed data by one-way ANOVA with a Bonferroni post-test. b Relative (pie chart) and absolute (bar chart) frequencies of nucleotide changes detected in rpoB of rifR clones from the indicated strains. The number of sequenced rifR is given in the center of each pie chart. c Location and relative frequency in % of mutated nucleotides in rpoB found in empty (blue), tet-dinB1Msm (red) or tet-dinB1Mtb+5aa (orange) rifR. d Absolute frequency of the main rpoB mutations found in indicated strains. confers tolerance to agents that damage template DNA. We, therefore, investigated the role of mycobacterial TLS polymerases in DNA damage tolerance. A previous study did not identify a role for Mtb dinB1 and dinB2 in damage tolerance38 but the possibility of redundancy between dinBs and dnaE2 was not tested. The M. smegmatis ΔdnaE2dinB123 mutant was not more sensitive than the WT to H2O2 (Supplementary Fig. 4a) or ciprofloxacin (Supplementary Fig. 4b). As reported by Boshoff et al.13, we found that the ΔdnaE2 deletion increased the sensitivity to UV (Supplementary Fig. 4c) and mitomycin C (Supplementary Fig. 4d) but we did not observe an additive effect caused by ΔdinB123 deletion. Nature Communications | (2022)13:4493 The data above indicate that DinB1 catalyzes substitution mutations that confer resistance to antibiotics such as rifampicin by abolishing drug binding while maintaining the functionality of the essential antibiotic target. However, the diversity of mutational alterations of the chromosome that impact bacterial phenotypes includes not only substitutions, but chromosomal rearrangements, deletions, and FS mutations. Recently, FS mutagenesis has emerged as an important mechanism of genome diversification in mycobacteria43,44 but the agents of FS mutagenesis in mycobacteria are not known. Translesion polymerases can introduce FS mutations during lesion bypass1–3, which prompted us to query the role of DinBs and DnaE2 in FS mutagenesis. To detect −1 FS mutations, we created a reporter system in which the chromosomal leuD gene carries a 2-base pair deletion in the second codon (leuD−2), which confers leucine auxotrophy (Fig. 5a). Reversion of this mutation by −1 or +2 FS confers leucine prototrophy (leu+) which is selected on leucine free media. In WT cells, the reversion frequency was 5 leu+/108 CFU (Fig. 5b). Sequencing of leuD in leu+ colonies revealed a −1 deletion in a run of 3T in almost half the revertants (44%). The other half had +2 addition (10%), >2-nucleotide insertion (7%), >2-nucleotide deletion (5%), or no mutation in leuD (32%) (Fig. 5b and Supplementary Table 5). The expression of the inactive dinB1Mtb did not increase leu+ frequency, but expression of dinB1Msm or dinB1Mtb+5aa increased leu+ frequency 4- or 27-fold due to a dominant proportion of −1 FS in the homo-oligonucleotide run of 3T. We then investigated the ability of DinB1 to incorporate +1 FS mutations using a leuD reporter with a one nucleotide deletion in the second codon (leuD−1) (Supplementary Fig. 5a). In WT cells, the leu+ frequency was 1/107 CFU but the mutations were mixed between +1 FS in leuD and an unexpected class of −1 FS mutations at the 3′ end of the upstream leuC gene, which suppressed the native leuC stop codon and restored the reading frame of the leuD coding sequence to create a 4 Article https://doi.org/10.1038/s41467-022-32022-8 101 100 H2O2 103 103 RifR/108 CFU RifR/108 CFU UV 104 ** 102 ** 102 MMS 103 RifR/108 CFU spont. 102 RifR/108 CFU a * 101 102 101 101 W T B1 Δd 2 na Δd 3 E 2 na d i E2 nB 12 3 in Δd W in T B1 Δd 2 na Δd 3 n E2 a di E2 nB 12 3 100 Δd W in T B1 Δd 2 Δ na d 3 E 2 na E di nB 2 12 3 Δd W in T B1 Δd 2 Δ na d 3 n E2 a di E2 nB 12 3 100 Δd 10-1 b n=29 A>T or T>A A>G or T>C A>C or T>G n=9 n=44 n=13 n=30 G>A or C>T G>T or C>A G>C or C>G n=14 60 4 400 2 200 40 40 20 0 n=32 80 other del no rpoB mut. 0 0 0 Δ W aE dn T 2d aE in 2 B1 23 n=29 MMS 120 Δd n n=39 Δ W a E dn T 2 d aE in 2 B1 23 n=50 H2O2 80 Δd n n=12 UV 600 Δ W aE dn T 2d aE in 2 B1 23 n=33 Spont. 6 Δd n MMS Δ W a E dn T 2 d aE in 2 B1 23 ΔdnaE2 dinB123 H2O2 Δd n ΔdnaE2 UV RifR/108 CFU WT Spont c 0.0 6.0 2.3 16.0 80 AGC CAG CTG TCG CAG TTC ATG GAC CAG AAC AAC CCG CTG TCG GGT 425 ..S ..Q ..L ..S ..Q ..F ..M ..D ..Q ..N ..N ..P ..L ..S ..G 18.2 2.3 20.0 4.0 2.3 2.3 4.0 2.0 36.4 18.0 2.3 0.0 0.0 2.0 CTG ACC CAC AAG CGT CGT CTT TCG GCG CTG GGC CCC ....... ACC 440 ..L ..T ..H ..K ..R ..R ..L ..S ..A ..L ..G ..P (+28aa) ..T 2.3 0.0 2.3 2.0 6.8 8.0 others no rpoB mut. 60 40 20 0 Δd n aE CCT GAG GGT CCC AAC ATC GGT CTG ATC GGT TCG 480 ..P ..E ..G ..P ..N ..I ..G ..L ..I ..G ..S 6.8 8.0 Other min. mut. Arg445(CGT>TGT) Ser438(TCG>TGG) Gln429(CAG>CTG) Leu427(CTG>CCT) Asn435(AAC>AAG) His442(CAC>GAC) His442(CAC>TAC) Ser438(TCG>TTG) Ser447(TCG>TTG) 2 4.5 0.0 0.0 2.0 2 H W 2O T 2 H 2 O 9.1 2.3 0.0 2.0 2.0 4.0 rpoB mut./108 CFU ΔdnaE2 H2O2: WT H2O2: Fig. 3 | DnaE2 but not DinBs mediates stress-induced substitution mutagenesis. a Rifampicin resistance (rifR) frequency in indicated strains and conditions. Results shown are means (±SEM) of data obtained from biological replicates symbolized by gray dots. Stars above bars mark a statistical difference with the reference strain (WT of each condition) (*, P < 0.05; **, P < 0.01). p-values were obtained on logtransformed data by one-way ANOVA with a Bonferroni post-test. b Relative (pie chart) and absolute (bar chart) frequencies of nucleotide changes detected in rpoB of rifR clones from the indicated strains. The number of sequenced rifR is given in the center of each pie chart. c Location and relative frequency of mutated nucleotides of rpoB found in rifR of ΔdnaE2 + H2O2 (orange) or WT + H2O2 (red). The bar chart shows the absolute frequency of the main rpoB mutations found in indicated strains. LeuC-LeuD fusion protein (Supplementary Fig. 5a). The expression of dinB1Msm increased leu+ frequency by 16-fold but the sequencing of leu+ colonies revealed that dinB1 expression exclusively catalyzed −1 FS at the 3′ end of the leuC gene, with 97% of these mutations in a 3C run. These results reveal that DinB1 can promote −1 FS mutations in the mycobacterial genome and does so more efficiently than promoting +1 FS mutations. mutagenesis in low complexity sequences. To more precisely measure the capability of DinB1 to incorporate −1 and +1 FS in homo-oligonucleotide runs in vivo and determine the effect of homomeric template sequence, we constructed integrative plasmids carrying a gene which confers resistance to kanamycin (kan) inactivated by the incorporation of 4T (kan::4T), 4C (kan::4C), 4G (kan::4G), 4A (kan::4A), 5T (kan::5T), 5G (kan::5G), or 5A (kan::5A) runs in the coding strand immediately 3′ of the start codon (Fig. 5a). These plasmids do not confer kanamycin resistance, but a deletion of one nucleotide (−1 FS) in the 4N run or +1 FS in the 5N run will restore the reading frame of kan allowing selection for kanR. DinB1 incorporates −1 FS and +1 FS in homo-oligonucleotide runs in vivo The location of the FS mutations in short homo-oligonucleotide tracts of leuC and leuD suggested that DinB1 may be a catalyst of FS Nature Communications | (2022)13:4493 5 Article https://doi.org/10.1038/s41467-022-32022-8 b MMS 150 *** 130 110 160 140 * 120 100 90 DinB1 can slip on homo-oligonucleotide runs in vitro M tb W T+ em +e pty +d m na pty +d E2 M in sm +d B 1 M in sm +d B 2 M in sm B +d 3 Ms in m +d B1 M in tb B2 Δd W in T B1 aE Δdn 23 2d aE in 2 B1 23 Δd n 85 ** 110 *** 90 ΔdnaE2dinB123 100 W Δd T in Δd B1 in Δd B2 Δd inB in 3 B Δd na Δd 123 E2 na di E2 nB 12 3 80 p=0.06 ** 140 *** 120 *** 100 80 M tb 120 ** 160 + pty +d em na pty +d E2 M in sm +d B1 M in sm +d B2 M in sm B +d 3 Ms in m +d B1 M in tb B2 p=0.05 Inhibition zone (WT%) p=0.06 em p=0.08 NFZ 180 T+ *** W *** p=0.06 *** f NFZ 180 130 M tb 100 Inhibition zone (WT%) 115 MNNG 150 W T+ em +e pty +d m na pty +d E2 M in sm +d B1 M in sm +d B2 M in sm B +d 3 Ms in m +d B 1 M in tb B2 *** ** W Δd T in Δd B1 in Δd B2 Δd inB in 3 B1 Δd na Δd 23 E2 na di E2 nB 12 3 Inhibition zone (WT%) d MNNG e Inhibition zone (WT%) * *** ΔdnaE2dinB123 130 140 * 80 c 160 kanR colonies of all strains. Expression of dinB1 increased the frequency of +1 FS localized in runs of 5T, 5G, and 5A by 3-, 6-, and 21-fold, respectively. dinB1Mtb+5aa expression also elicited +1 FS in the 5T (10-fold increase), 5G (3-fold increase), and 5A (5-fold increase) runs. Overall, these data reveal that mycobacterial DinB1 is a strong mediator of −1 FS and +1 FS in homo-oligonucleotide runs. MMS Inhibition zone (WT%) Inhibition zone (WT%) a ΔdnaE2dinB123 Fig. 4 | Redundancy of DinB1 and DnaE2 in tolerance to alkylation damage and N2-dG adducts. Sensitivities of indicated strains to a and b MMS, c and d MNNG, or e and f NFZ were measured by disc diffusion assay. Translesion polymerases were expressed under their native promoter from a genome integrated vector in panels b, d, and f. Results shown are means (±SEM) of data obtained from biological replicates symbolized by gray dots. Stars above the means mark a statistical difference with the reference strain (WT or ΔdnaE2dinB123 + empty in complementation experiments) (*, P < 0.05; **, P < 0.01; ***, P < 0.001). p values were obtained on log-transformed data by one-way ANOVA with a Bonferroni post-test. We first measured the ability of DinB1 to incorporate −1 FS in homo-oligonucleotide runs by using the kan::4N constructs. We found between 5 and 18 kanR/108 CFU, depending on the run sequence, in strains carrying the empty vector and the vast majority kanR colonies had −1 FS in the homo-oligonucleotide runs (Fig. 5c and Supplementary Fig. 5b, c, d). Expression of dinB1Msm enhanced −1 FS in runs of 4T, 4C, 4G, and 4A by 12-, 19-, 11-, and 3-fold, respectively. dinB1Mtb+5aa expression also increased −1 FS by 14- and 19-fold in 4T and 4C runs, respectively, but had no effect on runs of 4G or 4A. By using the kan::5N constructs, we quantified the capacity of DinB1 to incorporate +1 FS in homo-oligonucleotide runs. Between 10 and 74 kanR/108 CFU were detected in strains carrying the empty vector, depending on the nature of the run (Fig. 5d and Supplementary 5e, f). We found +1 FS in the run of the majority of the sequenced Nature Communications | (2022)13:4493 To test if the DinB1 polymerase is prone to slippage on homooligomeric template tracts in vitro, we employed a series of 5’ 32 P-labeled primer-template DNA substrates consisting of a 13-bp duplex with a 5′-tail composed of a run of four, six, or eight consecutive A nucleotides (A4, A6, A8) immediately following the primer 3′-OH terminus and flanked by three C nucleotides (Fig. 5e). Reaction of a DNA polymerase with the A4, A6, and A8 primer-templates in the presence of only dTTP should, if the polymerase does not slip or misincorporate dTMP opposite the template nucleotide following the A run, allow for the addition of four, six, or eight dTMP nucleotides to the primer terminus. However, if the polymerase is prone to backward slippage, then the primer strand can recess and realign to the template to allow one or more additional cycles of dTMP addition. Whereas most of the primer extension events catalyzed by DinB1 on the A4, A6, and A8 templates in the presence of dTTP did indeed cease at the end of the A run (e.g., denoted by ▸ for the A4 reaction in Fig. 5e), we consistently detected the synthesis of a minority product one nucleotide longer, consistent with a single slippage step mimetic of a + 1 frameshift (Fig. 5e). DinB1 displayed similar behavior when reacted with a series of DNAs in which the template strand tail comprised a run of four, six, or eight consecutive T nucleotides (T4, T6, T8) followed by three G nucleotides (Fig. 5e). In presence of dATP, the majority of the primer extension events on the T4, T6, and T8 templates entailed 4, 6, and 8 cycles of dAMP addition, respectively. +1 slippage products were also detected. The propensity to slip, defined as +1/[+1 plus ▸], increased progressively as the template T tract was lengthened from T4 (1%) to T6 (11%) to T8 (16%) (Fig. 5e). The +1 products are unlikely to have arisen via addition of a 3′-terminal mismatched dNMP, insofar as we could detect no extension of the 13mer primer stands on the A6 and T6 templates when DinB1 was presented with the incorrect dNTP (Supplementary Fig. 6). The finding that a DinB1 is capable of +1 slippage synthesis on a homo-oligomeric tract when the only dNTP available is that templated by the homo-oligomer does not reflect the situation in vivo where the polymerase will have access to the next correctly templated dNTP. To attempt to query whether provision of the next templated dNTP in vitro suppresses slippage, we included a dideoxy NTP (ddNTP): either ddGTP templated by the run of three C nucleotides following the A4, A6, and A8 tracts or ddCTP templated by the run of three G nucleotides flanking the T4, T6, and T8 tracts. ddNTPs are employed to force termination upon incorporation of the first templated nucleotide following the homo-oligomeric tract. Inclusion of the next templated ddNTP following the homo-oligomeric template tract suppressed the single slippage step mimetic of a + 1 frameshift observed in absence of ddNTPs (Fig. 5e), suggesting that +1 slippage by DinB1 is dampened by the presence of the next correctly templated dNTP. DinB1 was, in presence of ddNTPs, only partially effective in triggering conversion of the 17, 19, and 21 nt species to the respective ddG- or ddC-terminated 18, 20, and 22 nt products (Fig. 5e). We considered several possibilities, including: (i) that DinB1 might disengage from the primer-template when the 5′ tail comprising the template strand becomes shorter, and hence lose efficiency in adding opposite the third nucleotide from the 5′ end of the template strand; or (ii) DinB1 is inherently feeble at using dideoxy NTPs as substrates; or (iii) DinB1 and the primer 3′-OH end can slip forward on the template run by a single nucleotide on the homo-oligonucleotide run 6 Article https://doi.org/10.1038/s41467-022-32022-8 b leuD-2 leuD-2 TTTT AAAA .... AGC CAT ATT CAA CGG GAA ACG TCG GTA TAA GTT GCC CTT TGC ..s ..h ..i ..q ..r ..e ..t 150 *** * 10 1 d tetdinB1Msm tetdinB1Mtb+5aa n=28 n=9 n=17 no kan mut. tb + te Em t p te -din ty t-d B in 1 Ms B1 m M 5a a tb + te Em t-d pt y i te nB1 t M te -din sm t-d B in 1 Mtb B1 M 100 5T > +2 > -2 no leuD mut. 50 0 tb aa tb +5 te Em t p te -din ty t-d B 1 M in B1 sm M aa +5 tb te Em t p te -din ty t-d B in 1 Ms B1 m M Empty -1 in run -1 +2 100 10-1 0 -1 in run 101 te Em t p te -din ty t-d B in 1 Ms B1 m M 10-1 tetdinB1Mtb+5aa aa 20 n=12 p=0.09 KanR/108 CFU 40 tetdinB1Msm 150 aa 100 60 KanR/108 CFU 101 4T *** 102 n=26 e kan::5T 80 *** *** KanR/108 CFU KanR/108 CFU 102 Empty +5 kan::4T tb c AGC CAT ATT CAA CGG GAA ACG TCG GTA TAA GTT GCC CTT TGC ..s ..h ..i ..q ..r ..e ..t +5 TTTTT AAAAA ..... te Em t p te -din ty t-d B in 1 Ms B1 m M ATG TAC ..m 50 0 0.1 kan::5T n=41 100 5a a ATG TAC ..m kan::4T 100 Leu+/108 CFU ATG GA GAG GCT T TT TTC C ACC ACT CAC ACC GGC ATC CT C CGA A AA AG TGG TGA GTG TGG CCG TAG TAC C CTC AAG ..m ..e ..a ..f ..t ..t ..h ..t ..g ..i Leu+/108 CFU a Empty tetdinB1Msm tetdinB1Mtb+5aa n=17 n=9 n=16 +1 in run no kan mut. Fig. 5 | DinB1 promotes −1 and +1 frameshift mutations in homooligonucleotide runs. a leuD and kan frameshift (FS) reporter assays. leuD and kan open reading frame N-termini in which 2 base pairs in the second leuD codon were removed (blue box) or 4T/5T runs (red box) were incorporated upstream the start codon of kan. Reversion can occur by FS mutations that restore the leuD or kan reading frames resulting in phenotypic leucine prototrophy (leu+) or kanamycin resistance (kanR). The red box in leuD shows the run of 3T in which the majority of detected FS in leu+ were found. b leu+ and c, d kanR frequencies in the indicated strains in presence of inducer. Results shown are means (±SEM) of data obtained from biological replicates symbolized by gray dots. Stars above the means mark a statistical difference with the reference strain (empty) (*, P < 0.05; ***, P < 0.001). p-values were obtained on log-transformed data by one-way ANOVA with a Bonferroni post-test. Relative (pie chart) and absolute (bar chart) frequencies of nucleotide changes detected in leuD of leu+ cells or in kan of kanR cells represented with colors: red = −1 FS in the b 3 T run, c 4 T run, or d 5 T run, other colors = FS outside of the run, and gray = no detected mutation. The number of sequenced leu+ or kanR colonies is given in the center of each pie chart. e DinB1 polymerase reaction mixtures containing 5’ 32P-labeled primer-template DNAs with A4, A6, A8, T4, T6, or T8 runs in the template strand (depicted below and included as indicated above the lanes) and 125 µM deoxynucleotides and dideoxynucleotides (as specified above the lanes) were incubated at 37 °C for 15 min. DinB1 was omitted from reactions in lanes –. The reaction products were analyzed by urea-PAGE and visualized by autoradiography. The +1 slippage products are indicated. (mimetic of a −1 FS) and this species is extended by ddNMP incorporation to yield a product that comigrates with the 17, 19, and 21 nt species. These issues were addressed by reacting DinB1 with the A6 and T6 primer templates in the presence of various nucleotide substrates and combinations thereof. DinB1 catalyzed six steps of dTMP addition to the A6 template in the presence of dTTP and inclusion of dGTP elicited three further steps of dGMP addition opposite the 5′terminal CCC element of the template strand (Supplementary Fig. 6), indicating that DinB1 is competent for fill-in synthesis. In the reaction with ddTTP only, a small fraction of the primer was elongated by the expected single nucleotide step showing that DinB1 is, as hypothesized in scenario (ii), unable to efficiently utilize ddTTP as a substrate for correct templated addition. Similar results were obtained for the T6 primer template (Supplementary Fig. 6). The question remains whether any of the residual 17, 19, and 21 nt species seen in Fig. 5e represent −1 slips consistent with scenario (iii) above. In the case of the T tract templates, we see that the fraction of products that fail to be extended in the presence of ddCTP, defined as unextended product/[unextended product plus extended product], increases progressively as the template tract lengthens from 4T (13% unextended) to 6T (34%) to 8T (48%) (Fig. 5e). This result suggests a contribution of −1 slippage (rather than pure failure to incorporate ddC), the reasoning being that lengthening the template homo-oligonucleotide run is expected to enhance slippage but not impact DinB1’s ability to incorporate ddNTPs. Nature Communications | (2022)13:4493 DinB1 is the primary mediator of spontaneous −1 FS in runs of homo-oligonucleotides To determine the relative contribution of TLS polymerases in spontaneous FS mutagenesis, we measured the frequency of −1 FS in strains lacking dnaE2 or dinBs using the leuD−2 reporter (Fig. 6a). The ΔdinB2, ΔdinB3, and ΔdnaE2 deletions did not reduce the leu+ frequency or the proportion of −1 FS detected in leu+ colonies (Fig. 6a and Supplementary Table 5). In contrast, the leu+ frequency decreased by 5-, 3-, 7 Article https://doi.org/10.1038/s41467-022-32022-8 a leuD-2 4 WT ΔdinB2 ΔdinB3 ΔdnaE2 3 n=82 n=32 n=42 n=40 ΔdinB1 ΔdinB123 ΔdnaE2 dinB123 n=28 n=49 n=34 ** *** *** 1 0.1 2 1 W Δd T in B Δd 1 in B2 Δd Δd inB in 3 B1 Δd 2 Δ na dn 3 E2 aE di nB 2 12 3 0 c leuD-2 6 n=33 Leu+/108 CFU Leu+/108 CFU 10 WT +empty 4 2 > +2 subst. no leuD mut. kan::4C 8 100 n=29 ΔdinB1 +empty KanR/108 CFU * 1 -1 in run -1 > -1 +2 10 ** 1 *** *** KanR/108 CFU W Δd T in Δd B1 in B Δd 2 Δd inB in 3 B1 Δd 2 na Δdn 3 E2 aE di nB 2 12 3 b Leu+/108 CFU Leu+/108 CFU 10 WT 6 n=22 4 ΔdinB123 2 n=7 -1 in run -1 +2 > +2 subst. no leuD mut. 0 Δd W in T B1 23 n=35 0.1 W Δd T in Δd B 1 in B Δd 2 Δd i n B in 3 B1 Δd 2 Δ na dn 3 E2 aE di nB 2 12 3 0 W Δd T+ in em B Δd 1+ pty in em B1 p +d ty in B1 W Δd T+ in em B Δd 1+ pty in em B1 p +d ty in B1 0.1 ΔdinB1 +dinB1 -1 in run no kan mut. Fig. 6 | DinB1 is the primary mediator of spontaneous −1 frameshift mutations in homo-oligonucleotide runs. a, b leu+ or c kanR frequency in the indicated strains and relative (pie chart) or absolute (bar chart) frequencies of nucleotide changes detected in leuD or kan coded by color. The number of sequenced leu+ or kanR colonies is given in the center of each pie chart. Results shown are means (± SEM) of data obtained from biological replicates symbolized by gray dots. Stars above bars mark a statistical difference with the reference strain (WT) and lines connecting two strains show a statistical difference between them (*, P < 0.05; **, P < 0.01; ***, P < 0.001). p-values were obtained on log-transformed data by one-way ANOVA with a Bonferroni post-test. and 5-fold in the ΔdinB1, ΔdinB123 and ΔdnaE2dinB123, respectively, and the proportion of −1 FS mutations localized in the 3T run of leuD was also reduced. Expression of dinB1 in the ΔdinB1 strain restored both the WT leu+ frequency and the proportion of −1 FS localized in the 3T run of leuD (Fig. 6b and Supplementary Table 5). We extended these findings using the kan::4N reporters (Fig. 6c and Supplementary 7a, b, c). Compared to WT, the kanR frequency measured with the kan::4C reporter decreased by 9-, 9-, and 14-fold in the ΔdinB1, ΔdinB123, and ΔdnaE2dinB123 mutants, respectively, but we did not detect decrement in the ΔdinB2, ΔdinB3, and ΔdnaE2 mutants (Fig. 6c). The absolute frequency of −1 FS detected in the 4C run was reduced 54-fold in the ΔdinB123 mutant. In contrast, there was no impact of dinB1 deletion on the frequency of −1 FS mutation in 4T, 4G, or 4A runs (Supplementary Fig. 7a, b, c). In ΔdinB123 cells, −1 FS in the 4T run was reduced 4-fold but was unaffected in the 4G and 4A runs. Finally, we did not detect a significant impact of the TLS polymerase deletions on spontaneous +1 FS mutagenesis using the kan::5T, kan::5G or kan::5A reporters (Supplementary Fig. 7d, e, f). These results show that: (i) DinB1 is the dominant TLS polymerase involved in spontaneous −1 FS mutations in some homooligonucleotide runs; (ii) there is a redundancy between DinB1 and at least one other DinB for certain homopolymeric sequences; and (iii) endogenous levels of DinBs do not mediate spontaneous +1 FS mutations in unstressed cells. indicating that FS mutagenesis may be enhanced by DNA damage. To query the role of DNA damage in stimulating FS mutagenesis and the role of TLS polymerases in this process, we used the leuD and kan systems in conjunction with UV treatment. UV irradiation increased the frequency of leu+ in the WT strain carrying the leuD−2 reporter by 9-fold due to the induction of three main mutation types: −1 FS in the 3T run, −1 FS outside of the run, and base substitutions that in many cases created a new in-frame translational start codon that restored LeuD (Fig. 7a and Supplementary Table 5). The induction of the three mutation types was reduced in ΔdnaE2 and ΔdnaE2dinB123 mutants. The residual UV-dependent increase of the −1 FS mutations in the leuD 3T run in the ΔdnaE2 mutant was completely abolished in the ΔdnaE2dinB123 mutant, suggesting redundancy between DnaE2 and DinBs for damage-induced frameshifting. We also measured the impact of UV on −1 FS using the kan reporters. In the WT strain carrying the kan::4T vector, irradiation increased the frequency of kanR by fivefold and 100% of the sequenced clones had a −1 FS in the homo-oligonucleotide run (Fig. 7b). The −1 FS frequency was not reduced in either the ΔdnaE2 or ΔdinB123 mutants but decreased fivefold in the ΔdnaE2dinB123 mutant. UV treatment also enhanced −1 FS in the 4C, 4G, and 4A runs in WT cells but not in the dnaE2 mutant (Supplementary Fig. 8a, b, c). 25% and 43% of the DnaE2dependent mutagenic events detected with the kan::4G and kan::4A reporters, respectively, comprised −1 FS mutations located outside of the homo-oligonucleotide runs (Supplementary Fig. 8b, c), showing that the frameshifting activity of DnaE2 is not restricted to homooligonucleotide runs. Finally, although we detected a DnaE2dependent increase of kanR frequency in strains carrying kan::5T, kan::5G. or kan::5A reporters (Supplementary Fig. 8d, e, f), these events DnaE2 is the primary mediator of UV-induced −1 FS mutagenesis Prior literature13 as well as our data above (Supplementary Fig. 3) show that some mycobacterial TLS polymerases are DNA damage inducible, Nature Communications | (2022)13:4493 8 Article https://doi.org/10.1038/s41467-022-32022-8 Spont 45 *** n=82 WT n=24 n=24 WT +UV n=24 n=18 ΔdnaE2 dinB123 +UV UV *** p=0.06 *** 10 1 0.1 Leu+/108 CFU 30 W U T V W U Δd T V n Δd aE 2 i U nB1 V Δd 23 di na nB E 12 2 3 V W T 0 Δd WT n Δd Δ aE na din 2 E2 B1 di 23 nB 12 3 ΔdinB123 +UV ΔdnaE2 +UV 15 -1 in run -1 U Leu+/108 CFU 100 b leuD-2 kan::4T Spont 100 UV 10 ** * > +2 subst. no leuD mut. WT n=15 WT UV n=12 ΔdnaE2 UV 1 0.1 > -1 +2 n=23 *** W T Δd WT Δd Δ na na d i n E2 E2 B di 1 2 3 nB 12 3 leuD-2 KanR/108 CFU a -1 in run +2 no kan mut. Fig. 7 | DnaE2 is the primary mediator of DNA damage-induced −1 frameshift mutations in homo-oligonucleotide runs. a leu+ or b kanR frequency in indicated strains/conditions and relative (pie chart) or absolute (bar chart) frequencies of nucleotide changes detected in leuD or kan coded by color. The number of sequenced leu+ or kanR colonies is given in the center of each pie chart. Results shown are means (±SEM) of data obtained from biological replicates symbolized by gray dots. Stars above bars mark a statistical difference with the reference strain (WT + UV) and lines connecting two strains show a statistical difference between them (*, P < 0.05; **, P < 0.01; ***, P < 0.001). p values were obtained on logtransformed data by one-way ANOVA with a Bonferroni post-test. were not due to FS mutagenesis and rather were nucleotide substitutions that created a new start codon. These results reveal that DnaE2 is a major contributor to −1 FS mutations in response to DNA damage and that these FS are not restricted to homo-oligonucleotide runs. We also found that the DinBs have a redundant role with DnaE2 in −1 FS mutagenesis in response to DNA damage which differs depending on the sequence context. This work, together with previous DnaE2 studies, suggests that TLS polymerases may be attractive drug targets to prevent the acquisition of antibiotic resistance in Mtb13,33. However, we recently showed that mycobacterial TLS polymerases contribute to antibiotic bactericidal action elicited by the genomic incorporation of oxidized nucleotides when the MutT system is depleted53. Thus, TLS inhibition might have salutary effects on resistance, while diminishing the bactericidal effects of some antibiotics, a balance that will need to be assessed. In addition to its role in substitution mutagenesis, we found that DinB1 is the primary mediator of spontaneous −1 FS mutagenesis whereas DnaE2 is involved in DNA damage-induced −1 FS mutagenesis. We demonstrated that DinB1 is prone to FS in homo-oligonucleotide runs and that the FS mutagenic activity of DinB1 is conserved between M. smegmatis and Mtb. We observed low frequency of DinB1 slippage on homo-oligonucleotide runs in vitro, contrasting with the significant increase of −1 and +1 FS frequency measured in vivo when DinB1 is expressed. Because eukaryal translesion polymerase fidelity can be impacted by the PCNA clamp54 and because we found here that DinB1 mutagenesis depends on its β clamp interaction, we speculate that slippage might be increased if the β clamp was tethering DinB1 to the template. FS mutations are an increasingly recognized source of genomic diversity in Mtb. Mycobacterial genomes have a similar frequency of nucleotide substitutions compared to other bacteria but a higher frequency of FS mutations in homo-oligonucleotide runs55. This can be explained by the absence of the conventional MutS/L mismatch repair in mycobacteria, functionally replaced by a NucS-dependent system which can correct substitution mutations but not indels56,57. A recent in silico analysis of 5977 clinical Mtb isolates established that indels appear every 74,497 bases in genic regions and that the most common indel is −1 FS (two times more frequent than +1 FS and 6-fold more frequent than any other indel)43. These indels are significantly enriched in homo-oligonucleotide runs. Two seminal recent studies44,58 demonstrated a link between Mtb antibiotic tolerance and homo-oligonucleotide FS mutagenesis. Specifically, FS mutations in a run of 7C in the glpK gene, which encodes an enzyme of the glycerol metabolism, was found to control antibiotic tolerance and colony phase variation. This reversible phase variation is based on two successive FS mutations: first a +1 FS in glpK conferring tolerance and second a −1 FS restoring the original open reading frame of glpK. Reversible gene silencing through frameshifting is not restricted to the glpK gene. For example, another reversible drug Discussion Two decades ago, Boshoff et al. highlighted the importance of DnaE2 in mutagenesis, DNA damage tolerance, and pathogenicity in Mtb13. Because prior attempts to deduce a function for DinBs failed to reveal any phenotype38, DnaE2 has been considered the only mycobacterial TLS polymerase mediating chromosomal mutagenesis. However, our studies support a significant revision to this view and implicate a network of translesion polymerases in chromosomal mutagenesis. We confirmed that DnaE2 catalyzes the acquisition of rifampicin resistance in response to DNA damage13. We found that DnaE2 has the ability to induce a spectrum of rpoB mutations, particularly S447L, S438L, H442Y, and H442D. The codons of these amino acids are conserved between M. smegmatis and Mtb and these mutations represent ~75% of the rpoB mutations detected in the sequenced rifR Mtb clinical isolates (Supplementary Tables 4, 6, and45). Here we report that DinB1 can induce rifampicin resistance through a mutagenic activity. In contrast to the diverse mutation spectrum of DnaE2, DinB1 confers rifR through a unique rpoB mutation (CAC>CGC; H442R). This mutation has been detected in rifR Mtb clinical isolates at a frequency between 0.8% and 5%, depending on the study45–51 (Supplementary Tables 4, 6). Our data indicate shared and sequence context specific roles for DinB1 and DnaE2 in substitution mutagenesis and antibiotic resistance. This shared role raises several questions about how DinB1 and DnaE2 cooperate or compete at the replication fork. Both DinB1 and DnaE2 interact with the β clamp, DinB1 directly and DnaE2 via the ImuB protein33,52. Whether these two proteins compete for the same binding site, are recruited based on the type of damage, or can occupy different sites on the β clamp remains to be determined. Our study shows that DinB1 confers rifampicin resistance when the gene is highly expressed. Transcriptomic studies reported a 4.5-fold induction of dinB1Mtb caused by rifampicin treatment of Mtb35 and a 12-fold induction of the gene in pulmonary TB compared to culture conditions37. Further experiments will be needed to test if DinB1 participates to rifampicin resistance acquisition during infection. Nature Communications | (2022)13:4493 9 Article resistance mechanism mediated by FS in the orn gene has recently been reported in Mtb59. Moreover, Gupta and Alland identified 74 events in the genome of Mtb clinical isolates designated as “frame-shift scars”: two FS mutations in the same gene that disrupt and subsequently restore the integrity of the gene43. These events have been found in 48 genes of Mtb and multiple scars were detected in the ESX-1 gene cluster encoding a secretion system important for Mtb virulence. Frequent frameshifting in homo-oligonucleotide runs of the ESX-1 gene cluster of Mtb clinical isolates has also been reported60. High FS incidence has also been found in PE-PPE genes43,60 encoding secreted proteins61. The Mtb genome contains around 170,000 runs of three homo-oligonucleotides or more62. We believe that our study indicates that TLS polymerases are major contributors to the mycobacterial genome plasticity and advance DinB1 and DnaE2 as the prime mediators of homo-oligonucleotide FS mutagenesis. In this study, we also discovered that dinB1 and dnaE2 have a redundant role in resistance to alkylation damage in M. smegmatis, which could explain the lack of phenotypes obtained with Mtb dinB mutants38. The ability of DinB to confer alkylation damage tolerance has been reported in E. coli8 and for Pseudomonas aeruginosa and Pseudomonas putida, taxa in which a DnaE2 homolog also plays a role63. Alkylation damage can be generated by exogenous and endogenous sources64,65. Most endogenous sources of alkylation damage in bacteria are produced by metabolic enzymes catalyzing nitrosylation66,67. Mtb is exposed to nitrosative stress during macrophage infection25,28. A recent study reported that the pathogenic bacterium Brucella abortus encounters alkylating stress during macrophage infection and that the alkylation-specific repair systems are required for long-term mouse infection68. In Mtb, the deletion of similar alkylation-specific repair systems causes sensitivity to alkylating agents but does not impact virulence69,70, suggesting a possible functional redundancy between alkylation-specific repair systems and the TLS polymerases. Future studies will be conducted to investigate the redundancy between DnaE2, DinBs, and the alkylation-specific repair systems in the tolerance to alkylation damage and Mtb survival during infection. In summary, we have discovered a role of mycobacterial TLS polymerases, in particular DinB1, in alkylation damage tolerance, genome plasticity, and antibiotic resistance. By exposing the capability of DinB1 and DnaE2 to incorporate FS in homo-oligonucleotide runs, our work reveals potential molecular actors of reversible gene silencing, a recently discovered mechanism linked to antibiotic tolerance and virulence in Mtb43,44,59. https://doi.org/10.1038/s41467-022-32022-8 amplified using M. smegmatis mc2155 genomic DNA as template and were cloned into pAJF067 digested with Nde1 using recombinationbased cloning (In-Fusion, Takara). For plasmids carrying the kan gene inactivated by run of homo-oligonucleotides, the kan gene was amplified by PCR using the pAJF266 vector as template. The amplified fragments were cloned into pDP60 digested with EcoRI using recombination-based cloning (In-Fusion). The absence of mutations in constructs was verified by DNA sequencing. Growth and cell viability Cells in exponential growth phase cultured without inducer were back diluted in fresh 7H9 medium supplemented with 50 nM of inducer (Anhydrotetracycline: ATc) to OD600 = 0.001. Growth was measured by monitoring OD600 for 48 h. When OD600 reached a value around 1, cultures were back diluted in fresh +ATc 7H9 medium to OD600 = 0.001 and measured values were multiplied by the dilution factor. For cell viability, +ATc 7H9 liquid cultures were collected by centrifugation, resuspended in –ATc 7H9 and serial dilutions were spotted and cultured on –ATc Difco Middlebrook 7H10 agar medium and incubated 48 h at 37 °C. The number of CFU was counted and the result expressed in number of CFU per Optical Density Unit (ODU: CFU in 1 ml of a culture at OD600 = 1). For growth on agar medium, cells were grown in log phase without inducer and 5 μl of serial dilutions were spotter on 7H10 medium supplemented with 0, 5, or 50 nM of ATc and incubated at 37 °C for 72 h. Construct of M. smegmatis strains Plasmids were introduced into M. smegmatis by electrotransformation. The construct of unmarked deletion mutants used in this study is detailed in Dupuy et al.53. Unmarked 1 bp and 2 bp deletions upstream of the start codon of leuD were incorporated in each strain using a double recombination reaction as described in Barkan et al.71 and using plasmids listed in Supplementary Table 2. Plasmids carrying kan genes inactivated by an homo-oligonucleotide run, listed in Supplementary Table 2, were introduced at the attB site of the M. smegmatis genome. Disc diffusion assay Bacteria were grown to exponential phase, diluted in 3 ml of prewarmed top agar (7H9, 6 mg/ml agar) to an OD600 of 0.01 and plated on 7H10. A filter disc was put on the dried top agar and was spotted with 2.5 µl of 10 M H2O2, 10 mg/ml cip, 500 µg/ml mitomycin C, 100% MMS, 100 mg/ml MNNG, 50 mg/ml 4-NQO, or 100 mg/ml nitrofurazone. The diameter of the growth inhibition zone was measured after incubation for 48 h at 37 °C. Methods Bacterial strains Agar-based assay Strains used in this work are listed in Supplementary Table 1. Escherichia coli strains were cultivated at 37 °C in Luria-Bertani (LB) medium. M. smegmatis strains were grown at 37 °C in Middlebrook 7H9 medium (Difco) supplemented with 0.5% glycerol, 0.5% dextrose, 0.1% Tween 80. Antibiotics were used at the following concentrations: 5 μg/ml streptomycin (Sm), 50 μg/ml hygromycin (Hyg). Bacterial cultures grown to exponential phase were diluted to an OD600 of 0.1. Serial dilutions were performed from 100 to 10−5 in 7H9 and 5 µl of each dilution was plated on 7H10 or 7H10 supplemented with 0.05% MMS. Pictures were taken after 3 d incubation at 37 °C. Plasmids Plasmids and oligonucleotides used in this study are listed in Supplementary Tables 2 and 3. Plasmids were constructed in E. coli DH5α. For the construct of complementation plasmids, ORFs together with their 5′ flanking regions (∼500 bp) were amplified by PCR using M. smegmatis mc2155 or M. tuberculosis Erdman genomic DNA as template. The PCR products were cloned into pDB60 digested with EcoR1 using recombination-based cloning (In-Fusion, Takara). For the constructs of dinB1 expression plasmids, ORFs were amplified using M. smegmatis mc2155 or M. tuberculosis Erdman genomic DNA and were cloned into pMSG419 digested with ClaI. For the leuD inactivation plasmid construct, the ∼500 bp regions flanking the deleted nucleotides were Nature Communications | (2022)13:4493 UV sensitivity assay Bacterial cultures in exponential growth phase were diluted to an OD600 of 0.1 and serial dilutions (5 µl) were spotted on 7H10 plates. The plates were exposed to UV radiation (wavelength = 254 nm) at doses of 0, 5, 10, 15, or 20 J m−2 using a Stratalinker 2400 UV Crosslinker (Stratagene). Plates were imaged after 3 d incubation at 37 °C. Immunblotting Cell lysates were prepared from 2 ml aliquots of a log-phase culture (OD600 of 0.4) withdrawn at 0 h, 4 h, or 24 h after ATc addition to the cultures. Cells were collected by centrifugation, resuspended in PBS buffer supplemented with 0.1% Tween 20 (PBST), lysed by incubation with 10 mg/ml lysozyme for 15 min at 37 °C, and treated with 100 mM dithiothreitol for 10 min at 95 °C. Proteins were separated by 10 Article electrophoresis in a NuPAGE™ 4–12%, Bis-Tris gel (Novex) and transferred to a PVDF membrane. Blots were blocked and probed in 5% Omniblot milk in PBST. Proteins on blots were detected using antiRpoB (Biolegend, Cat#663905, RRID: AB_2566583) or anti-RecA (Pocono Rabbit Farm & Laboratory) antibodies incubated for 1 h at 1:10,000 dilutions and secondary Horseradish peroxidase-antibodies (#62-6520 and #65-6120, Invitrogen) at 1:1000 dilution. Blots were imaged in iBright FL1000 (Invitrogen) after treatment of the membrane with Amersham ECL western blotting detection reagents (GE Healthcare) according to manufacturer’s instructions. https://doi.org/10.1038/s41467-022-32022-8 was scanned with a Typhoon FLA7000 imager and the relative distributions of individual extension products were quantified with ImageQuant software. RT-qPCR and RNA sequencing RT-qPCR experiments were conducted exactly as described in Adefisayo et al.32 whereas RNAseq results were obtained from the RNAseq raw data published in Adefisayo et al.32. When overexpressed, dinB1Msm and dinB1Mtb expressions were measured 6 h after ATc addition. Quantification and statistical analysis Mutation frequency determination Bacteria were grown to exponential phase in 7H9 medium from a single colony. In experiments with deletion mutants, cultures were back-diluted at an OD600 of 0.0005 in fresh medium and cultured for 24 h. For dinB1 expression experiments, cultures were back diluted at OD600 of 0.004 in fresh medium supplemented with 50 nM ATc and cultured in presence of the inducer for 16 h. Cells (OD600 ~0.5) were concentrated 20-fold by centrifugation and pellet resuspension and 100 µl of a 10−6 dilution was plated on 7H10 agar whereas 200 µl was plated on 7H10 with 100 µg/ml rif or 4 µg/ml Kan for the measurement of substitution mutations or FS mutations in homo-oligonucleotide runs, respectively. For the measurement of FS mutations in leuD, cells were cultivated in 7H9 medium supplemented with 50 µg/ml leucine and plated on 7H10 (200 µl) or 7H10 supplemented with 50 µg/ml leucine. For stress-induced mutagenesis, cells were treated with UV (10 J m−2) or H2O2 (2.5 mM) for 2 h, washed and incubated for 4 h at 37 °C in fresh medium. MMS (0.010%) was added to the cultures 4 h before plating. Mutation frequency was expressed by the mean number of selected colonies per 108 CFU from independent cultures. For each strain and condition, the number of independent cultures used to measure the mutation frequency is indicated by the number of gray dots in each bar of graphs. For the determination of the mutation spectrum, the RRDR of the rpoB gene in rifR colonies, the kan gene in kanR colonies or the leuD gene in leu+ colonies, was amplified and sequenced using primers listed in Supplementary Table 3. For each strain, sequenced colonies were picked among at least six biological replicates. Mutation spectra were expressed as relative frequency, percent of mutations types found in each strain or condition, or absolute frequency, number of mutation types per 108 CFU obtained by multiplying the relative frequency of the mutation by the rifR, leu+ or kanR frequency. In vitro DNA slippage assay Recombinant M. smegmatis DinB1 was produced in E. coli and purified as described previously40. Protein concentration was determined by using the Bio-Rad dye reagent with bovine serum albumin as the standard. A 5′ 32P-labeled primer DNA strand was prepared by reaction of a 13-mer oligonucleotide, 5′-dCGTGTCGCCCTTC, with T4 polynucleotide kinase and [γ32P]ATP. The labeled DNA was separated from free ATP by electrophoresis through a nondenaturing 18% polyacrylamide gel and then eluted from an excised gel slice. The primer templates for assays of DNA polymerase were formed by annealing the 5′ 32P-labeled 13-mer pDNA strand (SG-FS1) to a series of unlabeled template strands (SG-FS2-7) at 1:3 molar ratio to form the primertemplates depicted in Fig. 5c and Supplementary Fig. 6. Polymerase reaction mixtures (10 µl) containing 50 mM Tris-HCl, pH 7.5, 5 mM MnCl2, 0.125 mM dNTP or ddNTP as specified, 1 pmol (0.1 µM) 32 P-labeled primer-template DNA, and 10 pmol (1 µM) DinB1 were incubated at 37 °C for 15 min. The reactions were quenched by adding 10 µl of 90% formamide, 50 mM EDTA, 0.01% bromophenol bluexylene cyanol. The samples were heated at 95 °C for 5 min and then analyzed by electrophoresis through a 40-cm 18% polyacrylamide gel containing 7.5 M urea in 44.5 mM Tris-borate, pH 8.3, 1 mM EDTA. The products were visualized by autoradiography. Where specified, the gel Nature Communications | (2022)13:4493 One-way analysis of variance (ANOVA) and a Bonferroni post-test were performed using Prism 9 software (GraphPad) on ln-transformed data for all statistical analyses of this work except for growth and viability experiments for which a 2-ways ANOVA was used. Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article. Data availability All data generated in this study are presented in the Figures and Tables. Source data are provided with this paper. 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L. et al. Contribution of rpoB mutations to development of rifamycin cross-resistance in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 42, 1853–1857 (1998). 12 Article 52. Gessner, S. et al. The mycobacterial mutasome: composition and recruitment in live cells. https://doi.org/10.1101/2021.11.16. 468908 (2021). 53. Dupuy, P., Howlader, M. & Glickman, M. S. A multilayered repair system protects the mycobacterial chromosome from endogenous and antibiotic-induced oxidative damage. Proc. Natl Acad. Sci. 117, 19517–19527 (2020). 54. Maga, G. et al. 8-oxo-guanine bypass by human DNA polymerases in the presence of auxiliary proteins. Nature 447, 606–608 (2007). 55. Springer, B. et al. Lack of mismatch correction facilitates genome evolution in mycobacteria. Mol. Microbiol. 53, 1601–1609 (2004). 56. Castañeda-García, A. et al. A non-canonical mismatch repair pathway in prokaryotes. Nat. Commun. 8, 14246 (2017). 57. Castañeda-García, A. et al. 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Simple sequence repeats in mycobacterial genomes. J. Biosci. 32, 3–15 (2007). 63. Jatsenko, T., Sidorenko, J., Saumaa, S. & Kivisaar, M. DNA polymerases ImuC and DinB are involved in DNA alkylation damage tolerance in Pseudomonas aeruginosa and Pseudomonas putida. PloS One 12, e0170719 (2017). 64. Beranek, D. T. Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents. Mutat. Res. 231, 11–30 (1990). 65. De Bont, R. & van Larebeke, N. Endogenous DNA damage in humans: a review of quantitative data. Mutagenesis 19, 169–185 (2004). 66. Sedgwick, B. Nitrosated peptides and polyamines as endogenous mutagens in O6-alkylguanine-DNA alkyltransferase deficient cells. Carcinogenesis 18, 1561–1567 (1997). 67. Taverna, P. & Sedgwick, B. Generation of an endogenous DNAmethylating agent by nitrosation in Escherichia coli. J. Bacteriol. 178, 5105–5111 (1996). 68. Poncin, K. et al. Occurrence and repair of alkylating stress in the intracellular pathogen Brucella abortus. Nat. Commun. 10, 4847 (2019). 69. Durbach, S. I. et al. DNA alkylation damage as a sensor of nitrosative stress in Mycobacterium tuberculosis. Infect. Immun. 71, 997–1000 (2003). 70. Yang, M. et al. The ADA operon of Mycobacterium tuberculosis encodes two DNA methyltransferases for inducible repair of DNA alkylation damage. DNA Repair 10, 595–602 (2011). Nature Communications | (2022)13:4493 https://doi.org/10.1038/s41467-022-32022-8 71. Barkan, D., Stallings, C. L. & Glickman, M. S. An improved counterselectable marker system for mycobacterial recombination using galK and 2-deoxy-galactose. Gene 470, 31–36 (2011). Acknowledgements This work is supported by NIH (NIH grant #AI064693) and this research was funded in part through the NIH/NCI Cancer Center Support Grant P30CA008748. P.D. was supported in part by a « Jeune Scientifique » salary award from the French National Institute of Agronomic Science (INRA). We thank all Glickman and Shuman lab members for helpful discussions. We thank Jamie Bean for reanalyzing the RNAseq data. Author contributions P.D., S.G., O.A., S.S., and M.S.G. designed research; P.D., S.G., O.A., and J.B. performed research; P.D., S.G., O.A., S.S., and M.S.G. analyzed data; P.D. and M.S.G. wrote the paper, with input from S.S. Competing interests M.S.G. has received consulting fees from Vedanta Biosciences, PRL NYC, and Fimbrion Therapeutics and has equity in Vedanta biosciences. All other authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-022-32022-8. Correspondence and requests for materials should be addressed to Michael S. Glickman. Peer review information Nature Communications thanks Veronica Godoy, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Reprints and permission information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. 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) 2022 13 

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Review published: 05 March 2018 doi: 10.3389/fimmu.2018.00427 Plasma Cell Differentiation Pathways in Systemic Lupus erythematosus Susan Malkiel1†, Ashley N. Barlev1,2†, Yemil Atisha-Fregoso1,3†, Jolien Suurmond1*‡ and Betty Diamond1‡ Center of Autoimmune Musculoskeletal and Hematopoietic Diseases, The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY, United States, 2 Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, United States, 3 Tecnologico de Monterrey, Monterrey, Mexico 1 Edited by: Ralf J. Ludwig, University of Lübeck, Germany Reviewed by: Rudolf Armin Manz, University of Lübeck, Germany Giovanni Di Zenzo, Istituto Dermopatico dell’Immacolata (IRCCS), Italy *Correspondence: Jolien Suurmond jsuurmond@northwell.edu These authors contributed equally to this work. † These authors share senior authorship. ‡ Specialty section: This article was submitted to Immunological Tolerance and Regulation, a section of the journal Frontiers in Immunology Received: 11 January 2018 Accepted: 16 February 2018 Published: 05 March 2018 Citation: Malkiel S, Barlev AN, Atisha-Fregoso Y, Suurmond J and Diamond B (2018) Plasma Cell Differentiation Pathways in Systemic Lupus Erythematosus. Front. Immunol. 9:427. doi: 10.3389/fimmu.2018.00427 Plasma cells (PCs) are responsible for the production of protective antibodies against infectious agents but they also produce pathogenic antibodies in autoimmune diseases, such as systemic lupus erythematosus (SLE). Traditionally, high affinity IgG autoantibodies are thought to arise through germinal center (GC) responses. However, class switching and somatic hypermutation can occur in extrafollicular (EF) locations, and this pathway has also been implicated in SLE. The pathway from which PCs originate may determine several characteristics, such as PC lifespan and sensitivity to therapeutics. Although both GC and EF responses have been implicated in SLE, we hypothesize that one of these pathways dominates in each individual patient and genetic risk factors may drive this predominance. While it will be important to distinguish polymorphisms that contribute to a GC-driven or EF B cell response to develop targeted treatments, the challenge will be not only to identify the differentiation pathway but the molecular mechanisms involved. In B cells, this task is complicated by the cross-talk between the B cell receptor, toll-like receptors (TLR), and cytokine signaling molecules, which contribute to both GC and EF responses. While risk variants that affect the function of dendritic cells and T follicular helper cells are likely to primarily influence GC responses, it will be important to discover whether some risk variants in the interferon and TLR pathways preferentially influence EF responses. Identifying the pathways of autoreactive PC differentiation in SLE may help us to understand patient heterogeneity and thereby guide precision therapy. Keywords: systemic lupus erythematosus, autoantibodies, B cells, plasma cells, tolerance INTRODUCTION Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of pathogenic autoantibodies that target a variety of nuclear self-antigens, some of which cross-react with tissue antigens. These autoantibodies cause tissue inflammation and lead to organ damage in the kidneys, skin, and more. Presence of IgG ANA is a diagnostic feature Abbreviations: AID, activation-induced cytidine deaminase; BCR, B cell receptor; BM, bone marrow; CSR, class switch recombination; DC, dendritic cell; EF, extrafollicular; FDC, follicular dendritic cell; FO, follicular; GC, germinal center; ICOS, inducible T-cell costimulator; IFN, interferon; MHC, major histocompatibility complex; MZ, marginal zone; PB, plasmablast; PBMC, peripheral blood mononuclear cell; PC, plasma cell; pDC, plasmacytoid dendritic cell; SHM, somatic hypermutation; SLE, systemic lupus erythematosus; Tfh, T follicular helper; TLR, Toll-like receptor. Frontiers in Immunology | www.frontiersin.org 1 March 2018 | Volume 9 | Article 427 Malkiel et al. PC Differentiation Pathways in SLE for SLE and other systemic autoimmune diseases, and these antibodies have an important role in disease pathogenesis (1, 2). In contrast, IgM ANA are considered to be protective against autoimmunity. They can be present in healthy individuals and assist in the non-inflammatory clearance of cellular debris and inhibit responses induced by IgG ANA (3–5). Other isotypes include IgA and IgE, but the pathogenicity of these isotypes has been less well studied. Antibodies are secreted by plasma cells (PCs), which arise as a terminal differentiation step from B cells. Most of our knowledge of immune tolerance to nuclear antigens, and the break of tolerance in SLE patients, is derived from studies with B cell receptor (BCR)-transgenic mice and single cell studies in humans, where self-reactivity is usually censored in developing B cells prior to their achieving immunocompetence (6, 7). Autoreactive cells that escape these mechanisms often become anergic (8–10), a process that mitigates against these cells giving rise to high affinity IgG autoantibody-producing PCs. Plasma cells can arise through two pathways: through activation of B cells and direct differentiation in extrafollicular (EF) foci or through a germinal center (GC) response. Although traditionally pathogenic high affinity autoantibodies have been associated with the GC response, recent insights have implicated the EF pathway in SLE as well. We hypothesize that both pathways can contribute to production of SLE autoantibodies. Understanding the regulation of each pathway and how genetic risk alleles may preferentially target one or the other of these pathways will be the focus of this review. Different subtypes of PCs have been described, including plasmablasts (PB), pre-PC, early PC, short-lived PC, and longlived PC. These terms are sometimes used interchangeably or not clearly defined. The confusion in part derives from the original paradigm that the EF pathway only results in short-lived proliferating PBs, whereas the GC pathway was thought to result only in long-lived quiescent PCs (11). However, lifespan and proliferation can operate independently from each other, such that there are short-lived PCs which are not proliferating, and long-lived PCs from GC origin can proliferate prior to becoming quiescent (12–14). In addition, PC differentiation is a continuum where expression of canonical B cell markers [B220, CD19, major histocompatibility complex (MHC) class II] is gradually lost and PC markers (such as Blimp-1, CD138, secreted Ig) are gradually upregulated (14). It is therefore difficult to define specific PC subsets based on the expression of these markers. Here, we define PCs as antibody secreting cells and we will only mention specific PC subsets if these have been clearly verified. Definitions used in this review are PBs, if proliferation has been verified; short-lived plasma cells, if a short lifespan of <7 days has been shown; or long-lived PC, if a long lifespan of >28 days has been demonstrated. of antigen. Antigens can activate B cells in a T-independent or T-dependent manner. T-independent responses do not require cognate T cell help. T-independent activation therefore leads to plasma cell differentiation in the absence of GCs. There are two types of T-independent antigens that can induce activation of B cells; TI-1 antigens can activate B cells through coengagement of Toll-like receptors (TLR), such as LPS or other bacterial polysaccharides, whereas TI-2 antigens lead to extensive crosslinking of the BCR, such as polymeric protein antigens or repeated structural motifs (15). In TI-2 responses, competition for antigen enhances the activation and expansion of high-affinity cells, while antigen affinity is less important in TI-1 responses (16). Although TI-1 and TI-2 antigens have been considered to induce T cell-independent responses, it is now clear that this distinction is not absolute: TI-2 and possibly TI-1 antigens can induce a transient GC (17), and the TI-2 serum antibody responses, in particular IgG, can still be T cell-dependent, even if the antigen cannot directly trigger T cells through MHC class II (18). It has therefore been proposed that the characteristics of the antigen is not the leading determinant of the response, but rather the B cell subset and the ancillary cell types involved determine the nature of the response (19). In addition to the strength of the initial stimulus through the BCR and cognate and non-cognate T cell interactions, B cell activation is also modified by the presence of other potent stimuli. Pattern recognition receptors, such as TLRs, interact with damageassociated molecular patterns or highly conserved microbial structures present in bacteria or virus. Included among these are both dsDNA (CpG enriched) and RNA. Many TLRs signal through MyD88, and MyD88-deficient mice have diminished antibody responses, both early and late after immunization (20–23). Simultaneous engagement of the BCR and TLRs has a synergistic effect on signaling and subsequent B cell activation (24). Cytokines, such as type I interferon (IFN), IL-6, and BAFF, can activate B cells and enhance both T-independent and T-dependent activation (25–27). BAFF has three recognized receptors, and one of them, TACI, signals through MyD88 (28), the same adaptor used by TLR7 and TLR9. Antigen-presenting cells, such as dendritic cell (DC) and macrophages, induce CD40-independent PC differentiation through secretion of cytokines such as BAFF and APRIL (26). Two processes that alter the antibody response are somatic hypermutation (SHM) and class switch recombination (CSR), both of which are mediated by the enzyme activation-induced cytidine deaminase (AID) (29). These processes can change antigen recognition by the BCR (SHM) or change the isotype that is expressed (CSR), and are usually associated with GC responses (discussed below). Although T-independent responses are usually associated with the IgM isotype, CSR can occur in certain infections and does not require cognate T–B interactions (30). CSR can be driven by MyD88 signaling or cytokines such as BAFF, APRIL, IFN-gamma, and IL-21 (20, 23, 31, 32). T-INDEPENDENT B CELL ACTIVATION AND PC DIFFERENTIATION B-1 Cells The B cell lineage consists of several subsets and cells diverge early in development. Each of these naive cell subsets can give rise to PCs, but they each preferentially respond to specific types Frontiers in Immunology | www.frontiersin.org B-1 cells represent a distinct population of B cells that arises during fetal development (33, 34). They are mainly found in the 2 March 2018 | Volume 9 | Article 427 Malkiel et al. PC Differentiation Pathways in SLE FO B Cells peritoneal and pleural cavities of mice and are rare in lymphoid organs and blood (Figure 1) (35). B-1 cells generally express germline-encoded, polyreactive IgM and IgA antibodies with limited V-gene segment usage, and are activated by T-independent antigens, such as LPS (TI-1) or multivalent antigens (TI-2) (36–38). In mice, B-1 cells can be further divided into B-1a and B-1b according to the expression of CD5 (CD5+ or CD5−, respectively). B-1a cells have been proposed as a major source of natural autoantibodies (37–40). These low-affinity polyreactive antibodies can be secreted spontaneously and are important in the clearance of apoptotic debris. They also contribute to protection against pathogens such as Streptococcus pneumoniae and influenza (41, 42). B-1b cells respond primarily to T-independent antigens (TI-1 and TI-2) and generate IgM memory cells, which contribute to protection against reinfection with Borrelia hermsii, S. pneumoniae, and Salmonella (19, 41, 43–45). B-1 cells are poor at forming GCs (46); however, class-switched, somatically mutated B-1 antibodies showing evidence of antigen selection have been isolated from humans (47). Although elevated numbers of B-1 cells are present in some lupus-prone mouse strains (36, 48), there is not a clear association with SLE (49, 50). Follicular B cells are migratory cells that move between lymph nodes, splenic follicles and the circulation until they interact with antigen. While MZ B cells are specialized in the response to T-independent antigen, they can also transport these antigens to the follicles and transfer such antigens to FO B cells (56, 57). However, compared to MZ B cells which become blasts within 24 h of mitogen activation, FO B cells do not show blast formation in response to mitogen, due to their requirement for cognate T cell help, and therefore the contribution of FO B cells in T-independent responses is probably limited (51). T-CELL-DEPENDENT ACTIVATION OF B CELLS Although there are models where T-independent responses can contribute to lupus in mice (58, 59), the majority of studies in lupus-prone mice and SLE patients suggest that T-dependent responses are the main driver of the disease. Therefore, we will focus on T cell-dependent responses for the remainder of this review. Here, we will first discuss the initial activation of B cells in a T-dependent response, including the cell fate decisions into either the EF or the GC pathway, followed by a discussion of the PC differentiation pathways after they diverge. T-dependent responses are thought to be dominated by FO B cells, although MZ B cells can migrate to the T–B border, activate T cells, and enter a GC (60–63). Although it is unclear how much they contribute to class-switched GC-derived antibody responses, MZ B cells can certainly contribute to T-dependent EF responses (Figure 2) (64, 65). MZ B cells can also capture and deliver blood borne antigens to the follicles, thereby enhancing T-dependent FO responses (66). This indicates that although MZ B cells do not require cognate T cell interactions for their differentiation into PCs, they can still participate in T-dependent responses (Figure 2). Marginal Zone (MZ) B Cells Marginal zone and follicular (FO) B cells differentiate from transitional B cells and both can participate in T-dependent and T-independent immune responses. MZ B cells are located in the MZ of the spleen, where they can serve as a first line of defense to T-independent and blood-borne antigens, such as lipopolysaccharide from bacteria (51). They are characterized by high responsiveness to TLR activation, as well as a preactivated state with high expression of complement receptors and costimulatory molecules. Due to these characteristics, they are known for their ability to quickly differentiate into PCs in response to T-independent antigens (51–53). MZ B cells do not require cognate T cell help, as soluble factors such as cytokines and costimulation derived from DCs, neutrophils, iNKT cells, and T cells, can also lead to their activation, CSR, and differentiation into PCs (28, 31, 54, 55). Figure 1 | B cell subsets in T-independent plasma cell development. B-1 cells in the peritoneal and pleural cavities can produce antibodies of the IgM and IgA isotype, either spontaneously (B-1a) or in response to T-independent antigens (B-1a and B-1b). Marginal zone B cells produce mostly IgM in response to T-independent antigens. Frontiers in Immunology | www.frontiersin.org Figure 2 | B cell subsets in T-dependent plasma cell (PC) development. Both marginal zone B cells and follicular (FO) B cells can contribute to the extrafollicular (EF) PC response, whereas the germinal center (GC) response and subsequent PC differentiation is predominantly driven by FO B cells. 3 March 2018 | Volume 9 | Article 427 Malkiel et al. PC Differentiation Pathways in SLE Recirculating FO B cells are activated by antigen in peripheral tissues or in lymphoid tissue where they encounter soluble antigen or antigen arrayed on follicular dendritic cells (FDCs). Activated B cells upregulate chemotactic factors (CCR7, EBI2) that favor their migration to the B–T border in the lymph nodes or spleen (67, 68). At the same time, CD4+ T cells are activated by DCs in the T cell zone, and these activated T cells will also migrate to the B–T cell border. The differentiation of Th cells [Th1, Th2, Th17, T follicular helper cells (Tfh)] is determined by T cell–DC interactions and is driven by the engagement of pattern recognition receptors by pathogens- or damage-associated molecules (69). At the T–B border, cognate interactions between antigen-specific B and T cells drives initial proliferation, and some B cells will undergo immunoglobulin CSR under the influence of T cell-derived cytokines (60, 70–73). After activation, B cells can diverge into EF PCs and GC B cells. While it is well known which transcription factors drive the differentiation into GC B cells versus EF PCs, whether there is direct competition between EF and GC differentiation has not been fully demonstrated. Since B cells can proliferate at the T–B border prior to making cell-fate decisions, it is possible that the distinction between cells with the same high affinity that enter an EF or a GC pathway is partly stochastic and that the same B cell clone can be found in both pathways (74, 75). Under non-competitive conditions, low-, medium-, or highaffinity B cells can all seed a GC, whereas the low-affinity cells are unable to generate an EF antibody response (76), due to failure to expand low-affinity EF PCs rather than a lack of initiation of PC differentiation (75). In contrast, under competitive conditions, low-affinity cells compete with high affinity cells and are unable to expand or enter a GC response (77). The advantage of high affinity B cells in each response can be at least partly explained by the degree of T cell help that is received, as high affinity B cells stimulated with antigen express higher amounts of MHC class II in the membrane, and are able to present more peptide to T cells, establishing a more effective immunological synapse (77), which can affect both EF and GC responses. Although TLR activation during T-independent responses clearly increases the magnitude of the EF response, TLR ligation in T-dependent responses can enhance both the GC response and the EF response (22), in a B cell-extrinsic or -intrinsic manner (78). lead to heavy chain CSR as well as initiation of PC differentiation (Figure 4). Under the influence of CXCR4, EF PCs migrate to the red pulp, where they can further proliferate and differentiate in EF foci. Proliferation is driven by BCR signaling, as cells with high affinity BCRs have increased proliferation and decreased apoptosis compared to cells with low affinity BCRs (75). Cofactors, such as CD19 and other molecules that enhance BCR signaling, can enhance EF proliferation. Lyn, an inhibitory molecule in the BCR signaling pathway, can diminish proliferation while in fact driving terminal PC differentiation (16). After the proliferative stage, PBs will differentiate further into PCs, characterized by higher expression of Blimp-1, further loss of MHC class II and costimulatory molecules. However, as discussed below, many EF PBs are short-lived and die prior to full differentiation into PCs (75). Some, however, complete their PC differentiation, after which they can survive in specialized niches in the spleen or bone marrow (BM) (12, 14). Recent evidence suggests that several characteristics previously attributed to GC responses can also occur in EF responses. This includes the formation of memory B cells, CSR, SHM, and induction of long-lived PCs (discussed below). Whereas the previous understanding was that memory B cells are generated only in the GC, memory cells initially appear in blood before GC formation (81), and Bcl-6-deficient mice, which are unable to form GCs, generate memory cells (82). Most of these are nonmutated and IgM + memory cells, suggesting an EF origin at least for some memory cells. Although CSR and a low degree of SHM occur during early B–T interactions at the T–B border, continued AID expression and affinity maturation in EF sites has been observed (83). GC Responses Germinal centers are areas of T-dependent B cell development in spleen, tonsils, lymph nodes, and Peyers’ patches. FDCs are important for normal splenic architecture and B cell development, as well as for maintaining the structure and function of the GC (84). Importantly, they capture antigen in immune complexes and retain the antigen in native form. Antigens are presented by FDCs on the cell surface (85). Several sequential events are involved in the formation of the GC. At the T–B border, B cell and T cell encounter with antigen stimulates formation of a GC (86), and migration from the T–B border into the follicle is mediated by CXCR5 (87). B cells receiving more T-help are more prone to differentiate into GC B cells (88, 89), and T cells differentiate into Tfh under the influence of B cell costimulatory molecules, including OX40L and CD80, which are essential for the maturation of Tfh cells (90, 91). The transcription factor Bcl-6 is required for the development of both GC B cells and Tfh cells (92). Tfh cells are specialized T helper cells that are involved in the selection and survival of B cells in the GC. The canonical costimulatory signal involved in the B–T cell interaction in the GC is CD40–CD40L (93, 94), but other signals such as ICOS–ICOSL, and IL-21 produced by Tfh cells are also required (95, 96). The structure of the GC, with a light zone and a dark zone, aligns with the processes of SHM, affinity maturation, and selection (Figure 5). B cells in the light zone are referred to as PC DIFFERENTIATION PC Differentiation in EF Responses As EF PCs can provide an initial wave of secreted antibodies during the first week of an infection, these cells are an important part of the initial antibody response against pathogens (11). During expansion at the T–B border, those cells destined to become EF PCs will upregulate Blimp-1, CD138, and CXCR4 (79). Differentiation of EF PCs is driven by T–B interactions with a specific subset of Th cells that resembles Tfh cells but is located in EF areas. These Th cells are dependent on Bcl-6 and Stat3, and their interaction with B cells is mediated through CD40-CD40L and inducible T-cell costimulator (ICOS)–ICOSL interactions as well as cytokines, such as IL-21 (75, 79, 80). These interactions Frontiers in Immunology | www.frontiersin.org 4 March 2018 | Volume 9 | Article 427 Malkiel et al. PC Differentiation Pathways in SLE centrocytes. They interact with FDCs through antigen and with Tfh cells through MHC–peptide interactions (97). Those B cells which make stronger interactions with Tfh cells, due to an increased T cell receptor peptide–MHC interaction, are positively selected and enter the dark zone where their proliferation is greater (98, 99). As more antigen is added, the population of B cells with BCRs that bind antigen sufficiently to induce positive selection increases (99). Positive selection in the light zone is important as it leads to GC B cells with the greatest affinity for antigen. Interactions between Tfh cells, antigen, and B cells in the light zone determine the extent of proliferation in the dark zone (99). Fewer cells move from the light zone to the dark zone than from the dark zone to the light zone indicating that selection occurs in the light zone (99, 100). The dark zone is the location where the most active proliferation of GC B cells takes place, as all GC B cells that are in G2 or M phase are in the dark zone; however, S phase cells are present in both the light zone and dark zone (100). Proliferation can occur under the influence of mTORC1 kinase, which activates the metabolic program that permits proliferation of B cells in the dark zone (98). After positive selection in the light zone and while undergoing proliferation in the dark zone, SHM occurs to effect a process called affinity maturation. During this process, point mutations occur in the BCR which affect its affinity for antigen. When the B cell returns to the light zone, the B cells that have undergone mutations to enhance affinity for the antigen are preferentially selected (101). A stronger interaction with Tfh cells in the light zone allows the B cell to undergo more rounds of proliferation in the dark zone. Therefore, each time the cell divides and more mutations are acquired, more affinity maturation can occur for B cells that were most positively selected for in the light zone (99). Negative selection also occurs in the GC. B cells with weak affinity for antigens in the GC, or autoreactive B cells recognizing ubiquitously expressed self-antigens are eliminated (102, 103). Proposed mechanisms for the negative selection of these B cells are Fas-mediated apoptosis of cells that fail to bind antigen, failure to receive continuing T cell help, or the activity of T follicular regulatory cells (Tfr) (102). A recent study, however, suggests that negative selection primarily occurs in cells with an unproductive BCR as a consequence of SHM rather than in cells with lower affinity (104). Certain cytokines favor the development of PCs. Among them, IL-21 is the most potent inducer of PC differentiation from memory and naive B cells (110, 111). This cytokine is produced by Tfh cells in the GC and activates the JAK1/3 STAT3 pathway. IL-21-deficient mice are unable to generate fully functional GCs. Without IL-21 or Tfh cells, PC formation is disrupted, affinity maturation does not occur, and the population of memory B cells is expanded (91, 96, 110). Toll-like receptor ligands also enhance GC responses through both DCs and B cells (21, 78, 112). Whereas soluble TLR ligands can enhance GC responses through an effect on DCs, an antigen that can trigger both endosomal TLRs and BCRs can enhance the IgG antibody response in a B cell-intrinsic manner (21). This probably reflects the requirement for BCR-mediated uptake of ligands for endosomal TLRs in this process, and explains why some studies reported no effect of TLR signaling on GC responses induced with LPS (113, 114). B cell-intrinsic MyD88 signaling specifically enhances the formation of GC B cells, affinity maturation, and CSR in response to the TLR-9 ligand CpG coupled to the hapten NP, without affecting the number of PCs. In contrast, MyD88 signaling in DCs contributes to PC differentiation without affecting affinity maturation (78). Different transcription factors are involved in the differentiation of PCs and memory B cells. Bach2 is reported to be important for selection of GC B cells into memory B cells; in the light zone, B cells with lower affinity for antigen have higher expression of Bach2, probably due to a lower degree of T cell help in those cells (105, 115). In addition to Bach2, ABF-1 leads to memory B cell differentiation and prevents PC differentiation (116). The transcription factors Blimp-1, XBP-1, and IRF4 are all involved in PC differentiation (117–119). Blimp-1 leads to decreased expression of genes involved in B cell signaling pathways including Pax5, which in turn leads to increased expression of Blimp-1 and XBP-1. This feed-forward mechanism is needed for PC differentiation (120, 121). Whereas Blimp-1 is required for PC differentiation, XBP-1 is more specifically needed for the unfolded protein response that is required for the production of high amounts of immunoglobulin in PCs (122). It has been recently reported that PC differentiation is initiated in light zone B cells after which they migrate to the dark zone to further differentiate (108). Together with simulation data, this suggests that PCs exit the GC through the dark zone (123). Similar to the EF response, GC-derived PCs are characterized by a proliferative PB stage. Proliferating PBs have been reported in the dark zone of the GC, as well as the T–B border directly adjacent to the GC (123, 124), and their proliferation decreases as they migrate further from the GC, and is completely lost as they reach the medulla of the lymph node or the splenic red pulp (124). This suggests that proliferation of GC-derived PBs occurs during their transit out of the GC, at distinct locations from EF PBs, which proliferate in EF foci mainly in the red pulp of the spleen or the medulla of the LN. Some GC-derived PCs migrate to the red pulp in the spleen or the medullary cords in the lymph nodes, and others migrate through the blood to the BM (14, 81). Their exit out of the secondary lymphoid organs occurs prior to completion of their differentiation, as circulating PBs that arise in PC Differentiation in the GC Both memory B cells and PCs arise from the GC, and many studies have examined the factors that determine if a given B cell will become a memory B cell or a PC. High affinity GC B cells become PCs, while lower affinity GC B cells become memory B cells (105–107). The initiation of PC differentiation in the light zone requires strong affinity for antigen; further differentiation in the dark zone requires help from Tfh cells (108). Light zone B cells become memory B cells early in the GC reaction, while PCs are formed later (105, 109). Preventing apoptosis in the GC allows for lower affinity B cells to become memory B cells but does not change the development of PCs, further suggesting that selection of B cells into the PC population is dependent on high affinity for antigen (106). Frontiers in Immunology | www.frontiersin.org 5 March 2018 | Volume 9 | Article 427 Malkiel et al. PC Differentiation Pathways in SLE GC responses in humans show signs of recent proliferation such as expression of Ki67 (14, 125). the time window for tolerance checkpoints. Therefore, it is likely that autoreactive B cells can be activated during EF responses, either through direct activation by self-antigen in an inflammatory milieu, cross-reactivity with foreign antigen, or through TLR ligands or cytokines (148). However, even if autoreactive PCs are generated in EF responses, they are mostly short-lived limiting the inflammation and tissue damage that is induced by autoantibodies. Therefore, the transient nature of the EF response may itself be a tolerance mechanism. In addition to the short-lived nature of the response, tolerance in EF responses can be maintained through the balance between IgM and other (more pathogenic) isotypes. As most EF PBs secrete IgM, even though some CSR can occur, the balance between IgG and IgM that is generated in EF responses may result in prevention of autoimmunity, through downregulating myeloid cell activation in a LAIR-1 dependent fashion and minimizing local inflammation (3, 5, 149). In addition, sialylation of IgG antibodies, which occurs in T-independent responses can also contribute to tolerance, as these antibodies have lower pathogenicity, at least in the context of rheumatoid arthritis [(150), p. 296; (151), p. 429]. T cell help during initial activation might play a role in tolerance in EF foci, where class-switched EF responses can occur through cytokines secreted by bystander T cells or non-T cells. Since there is no requirement for cognate T cell help, the T cell repertoire is unlikely to restrict autoreactivity in the EF response. PC Survival Two studies in the late 1990s showed the existence of long-lived PCs, disputing previous thinking that PCs were short-lived (126, 127). A more recent study showed that 10 years after vaccination, longlived PCs were still present in the BM, despite memory B cell depletion (128). Another study shows the survival of these long lived PCs despite CD19 directed CAR T cell therapy (129). These PCs have become a challenge in treatment of SLE, as they are often not eliminated by traditional therapies (130, 131). Although most evidence suggests that selection of PCs into the long-lived PC pool is dependent on extrinsic factors (132, 133), there is some evidence that B cell-intrinsic factors are also involved (134, 135). Identification of intrinsic factors leading to long-lived PC survival could represent therapeutic targets for SLE and other autoimmune diseases. Plasma cell survival depends on cytokines secreted by stromal cells and eosinophils in the BM (136, 137), but they can also survive in the spleen or other organs, particularly under inflammatory conditions. PCs can survive anywhere as long as sufficient survival factors are present (138), but niches have the capacity to support only a limited number of PCs (132). Two related factors important for survival of PCs are BAFF and APRIL, which act through binding to TACI and BCMA (139–141). Both cytokines are anti-apoptotic and increase PC survival (140). A study in autoimmune thrombocytopenia suggests that an increase in BAFF caused by B cell depletion promotes differentiation of short-lived PCs into long lived PCs in the spleen (142, 143). Other molecules which can enhance survival of PCs are IL-6, VCAM-1, CXCR4, and CD28 (11, 136, 144). CD93, a C1q receptor on B cells, is needed for the survival of PCs in the BM and is expressed only by a subset of PCs in mice (145). Induction of CD93 expression may, therefore, be an example of a B cell-intrinsic factor that contributes to PC survival. Despite the traditional paradigm mentioned above, there are descriptions of long-lived PCs in T-independent responses, T cell-deficient, and GC-deficient mice, with survival up to at least 100 days (12, 132, 146, 147). In addition, PCs exit the GC as PBs, and require a survival niche for full differentiation. As many of them fail to find the appropriate niche, not all GC-derived PCs are long-lived (12). As far as we know now, transcription factors that drive PC differentiation in each response are similar, and it is not clear if all PCs have the potential to become long-lived or whether some are selected, preferentially in the GC, to become long-lived, and whether this is accompanied by altered expression of key survival molecules and transcription factors that drive this distinction. Tolerance in GC Responses Although the mechanisms of central tolerance preclude many autoreactive B cells from entering GCs, self-reactive B cells developing in the BM can bypass tolerance mechanisms. This may occur if they are reactive to monovalent antigen, if their affinity for antigen is below a certain threshold, or if they are present in an inflammatory milieu (152, 153). Therefore, it is normal to have circulating autoreactive mature (naive) B cells (154). Importantly, however, mechanisms of peripheral tolerance are also in place to further eliminate autoreactivity. Mature selfreactive B cells can be thwarted from entering the follicle and be induced to become anergic (155). Still, some self-reactive B cells are able to enter the follicle. Evidence also suggests that autoreactive B cells that were initially excluded from the follicle can later be recruited into the GC, at which point these cells undergo SHM which may remove autoreactivity (156, 157), but can also lead to enhanced self-reactivity (153, 158). One important tolerance mechanism is the short lifespan of these B cells without mitogenic stimulation. Thus, only in an inflammatory milieu are these cells likely to access a GC response. B cells that acquire autoreactivity in the GC must be eliminated or prevented from becoming PCs. In the GC itself, several tolerance mechanisms have been described, including apoptosis and receptor editing. However, a recent study showed that autoreactive GC B cells are not strongly selected to undergo apoptosis, perhaps because so many autoreactive B cells are cross-reactive with an eliciting antigen (104, 159–163). It is conceivable that these tolerance mechanisms are initiated by lack of cognate T cell TOLERANCE Tolerance in EF Responses As autoimmunity has been traditionally thought to arise through the GC, tolerance checkpoints in EF responses have not been extensively studied. Whereas the fast rate of the EF response is needed for adequate responses against pathogens, it also limits Frontiers in Immunology | www.frontiersin.org 6 March 2018 | Volume 9 | Article 427 Malkiel et al. PC Differentiation Pathways in SLE help. As described, T-cell help is needed for positive selection of B cells into PCs in the GC, and without this help, self-reactive PCs will not develop (100, 102, 152, 153). Thus, although self-reactive memory B cells can develop, the requirement for Tfh cells and FDCs that recognize or present the autoantigen makes it more difficult for non-cross-reactive autoreactive PCs to develop. T cells recognizing foreign antigen may be able to stimulate autoreactive GC B cells, if the BCR crossreacts with the eliciting antigen or an antigen present in a multimolecular complex with the eliciting antigen. As IgG + memory B cells in healthy individuals have a much higher frequency of self-reactivity than IgG + PCs (164), an additional tolerance checkpoint must exist that prevents the differentiation of autoreactive PCs in addition to a tolerance checkpoint in GC B cells (Figure 3). Interestingly, switched PCs maintain expression of MHC class II and the antigen presentation machinery required for cognate T cell interactions at least until they are no longer proliferating, suggesting that this stage of PC differentiation may represent a T cell-dependent tolerance checkpoint (165). Although this has not been extensively studied, Th cells are required for the completion of GC-derived PC differentiation (103), and PCs can undergo cognate T cell interactions after their migration out of the GC at the T–B border (165). This suggests that a lack of T cell help may prevent the terminal differentiation of autoreactive PCs or that autoreactive B cells committed to becoming PCs are more susceptible to apoptosis or receptor editing than B cells committed to a memory pathway. addition, most studies discriminating EF responses from GC responses use acute immunization models, and it is not clear if all the paradigms that have been proposed for the distinction between EF and GC responses apply in the chronic immune activation present in autoimmune conditions. Although EF PC differentiation in autoimmunity has not been emphasized, recent studies indicate this pathway may have a specific role in autoimmunity (125, 166, 167). MRL/lpr mice exhibit EF PC generation, although they have increased formation of spontaneous GCs as well (166, 168, 169). In humans, recent research supports that a large proportion of the PCs in some SLE patients are clonally related to naive cells, suggesting an EF origin (125). Here, we propose mechanisms which can lead to enhanced EF responses in SLE (Figure 4). Expansion of MZ B Cells Marginal zone B cells are expanded in several lupus-prone mouse strains. In humans, the characterization of MZ B cells is much more complicated [reviewed in Ref. (13)], and it is unclear if SLE patients also have an expansion of this population. However, the high BAFF levels often present in SLE patients would support MZ expansion. Mice overexpressing BAFF develop an SLE-like phenotype that is characterized by a high titer of class-switched autoantibodies and PCs, in a T cell-independent manner (59). A preference for autoreactive B cells to differentiate into MZ B cells compared to FO B cells has been described in mice (170–173), and MZ B cells can differentiate directly into IgG + PCs in EF responses (174, 175). Therefore, development of serum autoantibodies in some lupus-prone mice has been attributed to MZ expansion and activation, although some studies have challenged this paradigm (176–180). PC DIFFERENTIATION IN SLE EF PC Differentiation in SLE Enhanced TLR Signaling Because there are no definite markers that discriminate PCs based on their pathway of differentiation, it is hard to establish the pathway through which they were derived, especially in humans where access to lymphoid organs is limited. In Another mechanism by which EF PC responses in SLE may be altered is through enhanced TLR signaling. MyD88-deficient MRL/lpr mice develop lower autoantibody titers and are Figure 3 | Tolerance in mature cell subsets in healthy individuals. B-1 cells are not shown in this figure as their relative autoreactivity compared to the other subsets is not exactly known. Marginal zone (MZ) B cells are enriched for autoreactive B cell receptors (BCRs) (polyreactive or antinuclear) compared to follicular B cells. IgG + memory B cells have a lower frequency of autoreactivity compared to naive B cells, suggesting a tolerance checkpoint in the germinal center. The autoreactivity in IgM plasma cells (PCs) has not been directly reported, but the fact that IgM autoantibodies are commonly found in healthy individuals and have an anti-inflammatory role, suggests that some autoreactive IgM + PCs are present and more common than autoreactive IgG + PCs. The low frequency of autoreactive BCRs in IgG + PCs compared to IgG + memory B cells suggests an additional strong tolerance checkpoint that prevents the development of serum IgG autoantibodies. Frontiers in Immunology | www.frontiersin.org 7 March 2018 | Volume 9 | Article 427 Malkiel et al. PC Differentiation Pathways in SLE Figure 4 | The extrafollicular (EF) pathway for the generation of autoreactive plasma cells (PCs) in systemic lupus erythematosus. Shown here are the potential mechanisms that can contribute to enhanced extrafollicular PC responses. These include (1) expansion of marginal zone (MZ) B cells, which often exhibit autoreactive receptors; (2) increased Toll-like receptor (TLR) signaling, which can directly activate B cells (B cell-intrinsic) or can enhance Th responses through their effect on dendritic cells (B cell-extrinsic); (3) B cell hyperresponsiveness, which can affect the activation of follicular B cells; (4) increased class switch recombination which can lead to more pathogenic IgG autoantibodies; (5) somatic hypermutation which can lead to affinity maturation of autoreactive PCs; and (6) increased PC survival. protected from disease (181, 182), suggesting a role for TLRs in EF responses in MRL/lpr mice. Although TLR-7 or -9 deficiency each diminished the production of specific types of autoantibodies in MRL/lpr mice, only TLR-7 deficiency diminished lymphocyte activation, IgG production, and kidney disease (183, 184). This suggests that although each receptor can enhance EF responses, only TLR-7 induces the production of pathogenic antibodies, or permits the inflammatory response needed to cause disease. Besides a B cell-intrinsic role of TLR signaling, B cell extrinsic TLR signaling can also enhance T–B interactions through the increased activation of DCs (185). Enhanced T–B interactions in this situation have the potential to enhance T-dependent PC differentiation in both EF and GC pathways. B cells as well as myeloid cells from SLE patients have increased expression of TLRs, and SLE patients may have increased proinflammatory responses to TLR ligands (186), which can contribute to stronger T-independent and T-dependent EF responses. genetic risk factors for SLE; discussed below) (189), or through a synergy between BCR triggering and other signaling pathways, such as TLR, BAFF, and type I IFN, which can each lower the threshold for B cell activation through the BCR and contribute to the activation of B cells (59, 190, 191). Type I IFN is necessary for a complete response after BCR/TLR7 stimulation, and increments in type I IFN can overcome tolerance that normally occurs after repetitive stimulation of TLRs (192). The fact that high affinity B cells are more prone to expansion at the EF PB stage (75, 76) suggests that the increased BCR signaling that occurs in SLE may preferentially stimulate EF responses. Increased CSR Increased CSR in EF responses is another feature of SLE that may contribute to enhanced pathogenicity of EF PCs, in particular if the balance between protective IgM and pathogenic IgG is altered. Increased CSR has been described both in lupus-prone mouse models as well as SLE patients (193). In particular, a special subset of EF T cells in the MRL/lpr mice has been described to contribute to the expansion of class switched IgG + EF PCs. These EF T cells are dependent on Bcl-6, Stat3, and ICOS, and they mediate IgG CSR through CD40–CD40L interactions and IL-21 (167, 168). A similar subset of T cells has been described in EF responses in B Cell Hyperresponsiveness A well-known feature of SLE is B cell hyperresponsiveness, which causes increased signaling upon BCR ligation by antigen (187, 188). The increased signaling can derive from increased activity of signaling molecules in the BCR pathway (many of which are Frontiers in Immunology | www.frontiersin.org 8 March 2018 | Volume 9 | Article 427 Malkiel et al. PC Differentiation Pathways in SLE non-autoimmune mice, although there they localized at the T–B border, and it is not clear if they migrate to EF foci as well (80). EF Th cells express CXCR4, as opposed to Tfh cells which express CXCR5 (or both) (194, 195). While the EF Th cell subset is present in MRL/lpr mice which have a dominant EF phenotype, mice with a more pronounced GC pathway, such as NZB/W, have a more mixed T cell phenotype (168). IgG CSR in MRL/lpr mice, as well as in graft versus host-mediated autoimmunity, is almost completely dependent on ICOS, as ICOS-deficiency leads to lower expression of CXCR4, as well as diminished secretion of IL-21 (167, 168). T-independent factors can also increase EF CSR in SLE. In AM14 rheumatoid factor transgenic MRL/lpr mice, T cells are required for the spontaneous production of rheumatoid factor, but not when B cells are exposed to chromatin immune complexes which will trigger both the BCR and TLR (196). Increased CSR has been described in circulating PBs of SLE patients, and at least some of these have low mutation rates, suggestive of an EF origin (125). However, all EF-derived PBs need not have low mutation rates. Factors that increase CSR in EF PCs in mice, such as IL-21, are increased in SLE patients (197), and factors that mediate T-independent CSR, such as TLR signaling and the myeloid-derived cytokine BAFF, are also increased in SLE (198). It is therefore conceivable that SLE patients can exhibit increased CSR in EF responses. to chromatin immune complexes in a T-independent manner (166, 168, 196, 199). This SHM potentially leads to affinity maturation (although probably to a lesser extent than in the GC) in autoreactive EF PCs, but no mechanism has been described for antigen selection and affinity maturation in EF responses. Increased SHM B Cell Hyperresponsiveness GC Responses in SLE Germinal center responses are well known to be increased in lupus-prone mice, and SLE patients have increased numbers of circulating pre-GC B cells, switched memory B cells and Tfh cells, suggestive of enhanced GC responses (169, 200, 201). Given that IgG anti-DNA autoantibodies which are considered to be pathogenic in SLE show evidence of SHM (202), the production of autoreactive PCs by SHM of nonautoreactive naive B cells within the GC has been considered an important contributor to the development of SLE in both mice (203) and humans (204). The following mechanisms can contribute to GC-derived autoreactive PCs (Figure 5). Loss of FO Exclusion In normal conditions, autoreactive naive B cells undergo anergy that leads to FO exclusion and prevents their recruitment into GC responses. However, in SLE, these B cells are able to enter the GC, and continue their differentiation into PCs (155, 205). Besides the increased CSR in EF PCs in MRL/lpr mice, SHM has also been shown to occur in EF foci, probably under the influence of EF Th cells. However, SHM can also occur in response As discussed above, B cell hyperresponsiveness can enhance EF responses and may also potentiate GC responses (206). Hyperresponsiveness could lead to increased positive selection Figure 5 | The germinal center (GC) pathway for the generation of autoreactive plasma cells (PCs) in systemic lupus erythematosus. Shown here are the potential mechanisms that can contribute to enhanced PC differentiation during GC responses. These include (1) loss of follicular exclusion, which can lead to recruitment of autoreactive B cells into GC responses; (2) B cell hyperresponsiveness which can affect the activation of follicular B cells; (3) increased Toll-like receptor (TLR) signaling, which can affect initial activation of B cells as well as the GC response itself; (4) de novo autoreactivity, generated through somatic hypermutation (SHM) and leading to the generation of autoreactive GC B cells from non-autoreactive precursors; (5) aberrant selection and survival, which can diminish tolerance mechanisms; (6) increased T follicular helper (Tfh) activity, which can increase the extent of GC responses as well as PC differentiation; (7) cell fate decisions that increase PC differentiation; and (8) increased PC survival. Frontiers in Immunology | www.frontiersin.org 9 March 2018 | Volume 9 | Article 427 Malkiel et al. PC Differentiation Pathways in SLE in the light zone and subsequent proliferation in the dark zone, thereby amplifying GC responses (207). differentiation of autoreactive B cells into autoreactive antibody secreting PCs (219). In lupus-prone mice, OX40L expression by B cells contributes to the autoimmune phenotype, presumably through its effect on Tfh cells (90, 220). Increased TLR Responses Toll-like receptors, which are involved in the EF pathway, also have a role in the GC response. Loss of MyD88 causes a loss of GC formation; interestingly, this alteration may be attributed to the function of TLR-7 and not TLR-9 (208, 209). Increased function of TLR-7 causes an increment in spontaneous GC formation and an autoimmunity phenotype (208). B cell-intrinsic MyD88 signaling specifically enhances GC responses when antigen and TLR ligand are coupled; self-antigen that can trigger both TLRs and the BCR will presumably have the same ability. It was recently demonstrated that FDCs, which are crucial for the maintenance of GCs and FO architecture, express type I IFN through a TLR-7 pathway upon internalizing complement-opsonized self-immune complexes through the complement receptor CD21 in the 564Igi RNP-specific lupus mouse model; this pathway is important for spontaneous GC formation and production of isotype-switched autoantibodies (210). 564Igi BM chimeras in which the recipient FDCs were TLR-7-deficient exhibited less autoimmunity. As many SLE antigens can activate TLRs (1, 190, 191), these represent potent pathways to amplify GC responses. Increased PC Differentiation B cell hyperresponsiveness may also increase the generation of PCs in SLE by directing more B cells to undergo PC differentiation. SLE patients often have increased numbers of circulating PBs; in one study this inversely correlates with the number of CD27 + memory cells, suggesting a preferred differentiation pathway (221). Lupus-prone mice, including the ones that have a GC phenotype, have vast increases in their PC numbers, which exceeds the expansion of the memory compartment. This suggests that there may be preferential output of PCs from the GC in SLE. Increased Survival of PCs Another possible mechanism for increased autoantibody titers is increased survival of PCs (Figures 2 and 3), which might occur if excess survival factors are present. Increased expression of the cytokines BAFF, APRIL, and IL-6 is present in lupus-prone mice (222, 223), suggesting that these cytokines can support enhanced PC survival. Although in healthy mice a limited number of PCs can survive in BM and spleen, these organs in lupus-prone mice gain additional capacity and exhibit an increased number of PCs (224). Both lupus-prone mice and SLE patients often exhibit hypergammaglobulinemia (225, 226), which may also be caused by an increased capacity to support PC survival in SLE patients. As both EF and GC-derived PCs can either stay in the spleen or stay in the lymph nodes or migrate to the BM, these factors will probably affect both types of PCs. In addition, several lupusprone mouse strains have increased levels of CXCL12 in their inflamed kidneys, which may allow recruitment of PCs to this organ (226–228). De Novo Autoreactivity Although loss of FO exclusion can lead to recruitment of autoreactive naive cells into GC reactions, SHM of nonautoreactive B cells can lead to de novo autoreactivity in GC B cells (202, 211). Whether these cells are able to differentiate into PCs has not been reported, but most pathogenic antibodies in SLE show signs of SHM, and de novo autoreactivity explains a large fraction of the autoreactive IgG + memory cells in SLE patients (204, 212). These studies suggest that GC B cells retain autoreactivity generated through SHM. Aberrant Selection and Survival of GC B Cells GENETIC RISK ALLELES Another mechanism for the generation of autoreactive PCs in the GC is increased survival of GC B cells. It has been shown that SLE patients have increased levels of BAFF (213). While BAFF has a large role in the EF pathway of differentiation of PCs, it also expands the Tfh cell population and promotes formation of GC and survival of B cells. This could be a contributing factor in allowing the breach of B cell tolerance seen in SLE patients (214, 215). As a result of increased BAFF, naive B cells with moderate affinity that would normally undergo apoptosis, may be rescued and enter a GC response. Whereas both EF and GC pathways can lead to autoantibody production, we propose that genetic factors may cause a dominance of either of these pathways in individual patients. Approximately one hundred risk loci have been associated with SLE, and genes within these loci have been broadly cast into categories involving DNA degradation and clearance of apoptotic/cellular debris, innate immunity, including TLR and IFN signaling, and adaptive immunity (229). Some overlap or fall outside these categories, and others are undefined. Most variants are in non-coding regions that may alter expression levels that can determine the magnitude of a response in a celllineage and stage-specific manner. Several risk genes have been described to alter B cell selection, activation, differentiation and/or survival in a B cell-intrinsic fashion, including LYN, BLK, BANK1, PTPN22, TNFAIP3, TNIP1, CSK, and FCGR2B (189, 230). Although it is known that these risk alleles alter B cell signaling, their effect on PC differentiation has not been Increased Tfh Activity Another mechanism that leads to the development of autoreactive PCs in the GC is increased Tfh activity (216). One cytokine important for the development of autoreactive PCs is IL-21, a cytokine produced by Tfh cells (72). The number of Tfh cells as well as the level of IL-21 has been shown to be increased in lupusprone mice and SLE patients (217, 218). IL-21 increases IgG PC number (72, 217), and Tfh cells can alter selection and allow the Frontiers in Immunology | www.frontiersin.org 10 March 2018 | Volume 9 | Article 427 Function of gene Function of risk allele Potential role in EF Potential role in GC HLA class II genes Antigen presentation Presentation of self-specific T cell epitopes May increase T-dependent EF responses Expansion of Tfh, GC responses and PC differentiation TNFS4 (OX40L) Costimulatory molecule on many cell types primarily interacting with OX40 on activated T cells promoting T cell functions, cytokine and Ab production, and PC generation Most likely a response eQTL, as DNA heterozygous for the 5′ rs2205860 SNP had enhanced binding to NF-κB; no significant differences in basal expression in EBV-transformed cells or primary cells Required for T cell-dependent EF Ab response driven by MZ DCs Supports Tfh maturation in mice (B cell-intrinsic) and in humans (expression on myeloid APCs) (64, 90, 220, 233, 234) CD80 T cell costimulation through CD28, CTLA-4, PD-L1 Unknown Little effect on Ab production by short-lived plasmablasts (in CD80−/− mice) Increased maturation of Tfh and generation of long-lived PC (91) LYN Src-family kinase that phosphorylates both activating and inhibitory receptors in B cells and myeloid cells. Its role in activating ITAMs is probably redundant with other Src family kinases, therefore its role in inhibitory receptors seems most crucial Unknown SLE patients have decreased expression of Lyn in B cells Lyn−/− mice have EF PC differentiation without GCs in some studies Lyn−/− mice have spontaneous GCs in some studies (187, 235, 236) BLK Src-family kinase that phosphorylates both activating and inhibitory receptors in B cells Decreased expression in B cells, increased B cell activation Increased TI IgG antibody responses in Blk+/− mice, with no effect on IgM Increased numbers of switched memory cells in risk carriers suggests more active GC responses, but TD antibody responses in Blk+/− or −/− mice not affected (237–240) BANK1 Signaling molecule involved in BCRand CD40-mediated signaling in B cells, positively regulates Ca2+ release in B cells, negatively regulates CD40-mediated signaling Differential expression of two splice variants, but functional consequences unknown Normal antibody responses to TI antigen, but the IgM response in TD responses was increased, possibly due to increased survival of EF PCs Normal IgG antibody responses to TD antigen suggesting that there is no major influence on switched TD responses, although there is a slight increase in spontaneous IgG2a in Bank1−/− mice (241–243) PTPN22 (Lyp) Protein tyrosine phosphatase that has inhibitory function in B and T cell signaling Risk allele has increased inhibitory function, causing decreased B cell activation, proliferation and signaling leading to impaired central B cell tolerance as well as impaired T cell responses Impaired central tolerance; unclear if PTPN22 affects EF responses; deficiency of PEP (mouse ortholog) in mice did not alter spontaneous IgM and IgG3 levels, suggesting no effect on extrafollicular antibody production Lower frequency of memory cells in risk allele carriers suggests that it may inhibit GC responses consistent with increased GCs and serum IgG in PEP−/− mice TNFAIP3 (A20) Negative regulator of NF-κB signaling in response to TLR, TNF, and CD40 signaling in B cells and other immune cells Reduced expression in EBV transformed cells with one risk variant and reduced anti-inflammatory activity in transfected HEK cells with another risk variant B cell-specific A20 deficiency in mice leads to alterations in the MZ compartment and consistently enhanced IgM production (spontaneous, as well as TD and TI immunizations), but no difference in IgG3 B cell specific A20 deficiency in mice leads to elevated numbers of GC B cells, and spontaneous IgG2 levels in old mice which deposited in kidneys; however, inconsistent effects on TD IgG production upon immunization in different studies 11 Gene Reference Malkiel et al. Frontiers in Immunology | www.frontiersin.org Table 1 | The role of genes with risk alleles in EF and GC responses. (103, 231, 232) (206, 244–247) (Continued) PC Differentiation Pathways in SLE March 2018 | Volume 9 | Article 427 (248–253) Function of risk allele Potential role in EF Potential role in GC Reference Ubiquitin-binding protein with diverse targets; interaction with TNFAIP3 negatively regulates NF-κB; also known to repress PPARs, which may increase B cell activity Reduced expression in EBVtransformed B cells from H1 and H2 risk haplotypes; H1 contains coding SNP near a nuclear export sequence Mutation of polyubiquitin binding site increased formation of EF PCs Mutation of polyubiquitin-binding site induced spontaneous GC, increased TFH, CSR, and production of autoreactive Abs thru TLR-mediated NF-κB pathway (254, 255) CSK Tyrosine kinase protein that phosphorylates Src family kinases leading to their inactivation. Src family kinases can act on both activating and inhibitory receptors in B and T cells Increased expression in B cells, increased B cell activation (Lyn phosphorylation, Ca2+ mobilization), expansion of transitional B cells Unknown, but increased signaling may enhance PC differentiation Csk is low in memory cells but its function in GC responses is not known (256) FCGR2B Inhibitory receptor for IgG on B cells and other immune cells Impairment of receptor mobility, lipid rafts and inhibitory signaling Enhanced antibody production upon TI immunization, although not observed in all studies Enhanced GC responses in FCGR2B−/− mice. Spontaneous GC B cells have increased self-reactivity, but the checkpoint to PCs is still intact in FCGR2B−/− mice, so uncertain if autoreactive PCs in these mice are GC-derived (150, 257–262) IRF5 Production of type I IFN in response to TLR ligands, macrophage polarization, enhanced PC differentiation Increased expression and activation in monocytes from SLE patients with the risk allele IRF5−/− mice have decreased IgG1 responses upon TI immunization and have decreased PC numbers in MRL/lpr mice, suggesting that increased expression of IRF5 may enhance EF responses IRF5−/− mice have diminished GC-derived antibodies, suggesting the IRF5 risk allele may enhance GC PC differentiation (263–267) STAT4 Transcription factor critical for myeloid and lymphocyte functions; major responder to IL-12; role in IFN-α signaling Increased expression in PBMCs correlated with SNPs rs3821236, rs3024866 (both in the same haplotype block) and rs7574865 but not with other SNPs STAT4−/− had no effect on antibody titers or pathology in EF model (MRL/lpr) Regulates Tfh through Bcl-6 and T-bet in T cells; indirectly upregulates T-bet in B cells, which facilitates spontaneous GC; STAT4−/− reduced autoantibody production and glomerulonephritis in B6.TC model (Sle1,2,3 congenic) (268–270) BACH2 Transcriptional repressor that promotes CSR/SHM and is required for memory B cell differentiation No expression differences associated with rs597325 in primary blood cell types Deficiency increases IgM PC differentiation in vitro Bach2 can enhance memory B cell differentiation while blocking plasma cell differentiation (105, 271–273) PRDM1 (Blimp-1) Transcription factor required for PC differentiation/transcription factor that alters DC function Decreased expression of Blimp-1 in DCs leading to increased cytokine production (unknown function of risk allele in PCs) Unknown Expansion of Tfh and increased GC responses in DC-Blimp-1-deficient mice (274, 275) IRF8 Transcription factor that inhibits PC differentiation together with PU.1 and distribution into FO or MZ compartments Increased expression in EBVtransformed cells Increased IRF8 expression presumably would decrease PC differentiation Increased function of IRF8 could lead to enhanced GC responses, through regulation of Bcl-6, AID, and MDM2 (276–278) IKZF3 (Aiolos) Transcription factor involved in lymphocyte development and function; important in B cell maturation and activation Unknown Deficiency impairs the MZ B cell compartment but not the generation of short-lived PCs following immunization Deficiency induces spontaneous GCs and production of autoantibodies; deficiency prevents generation of high-affinity BM PCs following immunization (279–281) March 2018 | Volume 9 | Article 427 (Continued) PC Differentiation Pathways in SLE Function of gene TNIP1 12 Gene Malkiel et al. Frontiers in Immunology | www.frontiersin.org TABLE 1 | Continued (290–293) T-dependent IgG2a/c OVA responses are decreased in SLC15a4−/− mice, but not in SLC15a4 mutant mice. IgG autoantibodies in pristane-induced lupus aC15a4 deficiency, suggesting that reduced expression in risk allele carriers may block GC-derived PCs SLC15a4 mutant mice, which are unable to produce IFN1, have reduced anti-chromatin IgG and IgM in B6/lpr mice, but have otherwise normal T-dependent and independent responses One risk variant is associated with reduced expression in monocytes Lysosomal amino-acid transporter required for endosomal TLR signaling and IFN1 production SLC15A4 Frontiers in Immunology | www.frontiersin.org extensively investigated, and most of our understanding of the role of these genes in EF and GC responses derives from mouse models. Other risk alleles are involved in B–T cell interactions, memory or PC differentiation, and IFN/TLR signaling. For many of these, the functional consequence of the risk allele has not been determined. However, it is reasonable to ask whether these risk alleles may alter EF or GC responses. Table 1 shows risk alleles that can alter B cell responses and subsequently PC differentiation. These risk alleles can function in a B cell-intrinsic or -extrinsic manner. We propose that some risk alleles, such as TLR7, FAS, IRF5, TNFAIP3, and TNIP1, can modify both EF and GC responses. Certain risk alleles, such as HLA class II genes, FCGR2B, STAT4, CD80, IRF8, and PRDM1, most likely drive GC responses, whereas other risk alleles, such as ETS1, LYN, BACH2, and BLK, may preferentially drive EF responses in SLE, although this pathway has not been extensively explored. Further understanding of the exact role of each risk allele in plasma cell differentiation pathways may enhance our insight into patient heterogeneity. CONCLUSION The categories “potential role in EF or GC” are speculative and based on our understanding of the literature included in the reference column. (184, 208, 210, 288, 289) GC responses are driven by B cell-intrinsic TLR7 in B6 and B6.Sle1b mice and FDCs in 564Igi RNP-specific lupus model EF PCs in AM14 Tg MRL/lpr mice are driven by TLR-7 Increased expression in PBMCs Activation of immune cells by RNA viruses or self-antigen TLR7 (286, 287) Fas/FasL interactions are important for apoptosis in GC, thereby potentially affecting survival and selection of autoreactive GC B cells Based on studies in MRL/lpr mice, increased expression of FasL would be expected to decrease EF PC responses Increased surface expression on circulating fibrocytes Apoptosis of immune cells through engagement of Fas FASL (282–285) Loss of anergy may increase participation of autoreactive B cells in GC reactions Increased EF PC responses in Ets1−/− Reduced expression of Ets1 in EBVtransformed cells and PBMCs Transcription factor important for lymphocyte development and differentiation; it is also involved in maintaining B cell tolerance through anergy maintenance ETS1 Reference Potential role in GC Potential role in EF Function of gene Gene TABLE 1 | Continued PC Differentiation Pathways in SLE Function of risk allele Malkiel et al. In this review, we have described the PC differentiation pathways which can contribute to the development of autoantibody production in SLE. Whereas both EF and GC pathways may be active in the same patient, we propose that certain genetic risk alleles contribute to the dominance of one of these pathways. The dominant PC differentiation pathway, determined by the composite of risk alleles, may contribute to patient heterogeneity and to response to therapy. Although it is likely that different therapeutics alter each pathway to a different extent, there is to our knowledge not enough understanding of the molecular pathways in each response nor is there clear evidence which therapeutics target which pathway. These pathways are not as distinct as we thought, nor can the pathway taken by a PC be easily distinguished with current knowledge. 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Interferon regulatory factor-5 deficiency ameliorates disease severity in the MRL/lpr mouse model of lupus in the absence of a mutation in DOCK2. PLoS One (2014) 9(7):e103478. doi:10.1371/journal.pone.0103478 Thackray LB, Shrestha B, Richner JM, Miner JJ, Pinto AK, Lazear HM, et al. Interferon regulatory factor 5-dependent immune responses in the draining lymph node protect against West Nile virus infection. J Virol (2014) 88(19):11007–21. doi:10.1128/JVI.01545-14 Abelson AK, Delgado-Vega AM, Kozyrev SV, Sanchez E, Velazquez-Cruz R, Eriksson N, et al. STAT4 associates with systemic lupus erythematosus through two independent effects that correlate with gene expression and act additively with IRF5 to increase risk. Ann Rheum Dis (2009) 68(11):1746–53. doi:10.1136/ard.2008.097642 Liang Y, Pan HF, Ye DQ. Therapeutic potential of STAT4 in autoimmunity. Expert Opin Ther Targets (2014) 18(8):945–60. doi:10.1517/14728222.2014. 920325 Xu Z, Duan B, Croker BP, Morel L. 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Slc15a4, AP-3, and Hermansky-Pudlak syndrome proteins are required for toll-like receptor signaling in plasmacytoid dendritic cells. Proc Natl Acad Sci U S A (2010) 107(46):19973–8. doi:10.1073/pnas.1014051107 Sasawatari S, Okamura T, Kasumi E, Tanaka-Furuyama K, Yanobu-Takanashi R, Shirasawa S, et al. The solute carrier family 15A4 regulates TLR9 and NOD1 functions in the innate immune system and promotes colitis in mice. Gastroenterology (2011) 140(5):1513–25. doi:10.1053/j.gastro.2011. 01.041 Kobayashi T, Shimabukuro-Demoto S, Yoshida-Sugitani R, Furuyama-Tanaka K, Karyu H, Sugiura Y, et al. The histidine transporter SLC15A4 coordinates mTOR-dependent inflammatory responses and pathogenic antibody production. Immunity (2014) 41(3):375–88. doi:10.1016/j.immuni.2014. 08.011 Baccala R, Gonzalez-Quintial R, Blasius AL, Rimann I, Ozato K, Kono DH, et al. Essential requirement for IRF8 and SLC15A4 implicates plasmacytoid dendritic cells in the pathogenesis of lupus. Proc Natl Acad Sci U S A (2013) 110(8):2940–5. doi:10.1073/pnas.1222798110 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. The reviewer RM and handling editor declared their shared affiliation. Copyright © 2018 Malkiel, Barlev, Atisha-Fregoso, Suurmond and Diamond. 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. 21 March 2018 | Volume 9 | Article 427 

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A B WT 1 2 Y537S Mutant D538G Mutant 3 1 2 3 1 2 Molecular and Cellular Functions [Shared Mutant Genes] Direction p-value −2 D538G allele specific Up n = 1207 Down n = 1201 p-value 4.77E-14 DNA metabolic process 4.58E-09 regulation of transcription by RNA polymerase II 4.36E-07 nuclear-transcribed mRNA catabolic process, nonsense-mediated decay 2.48E-12 DNA replication 1.19E-08 double-strand break repair via homologous recombination 4.56E-06 cellular protein metabolic process 4.89E-10 G2/M transition of mitotic cell cycle 5.44E-07 DNA replication 2.29E-05 aerobic electron transport chain 6.60E-10 G1/S transition of mitotic cell cycle 1.12E-06 microtubule cytoskeleton organization involved in mitosis 2.61E-05 mitochondrial ATP synthesis coupled electron transport 9.19E-10 double-strand break repair via homologous recombination 1.62E-06 DNA repair 5.74E-05 de novo' posttranslational protein folding 3.76E-07 regulation of transcription involved in G1/S transition of mitotic cell cycle 3.13E-06 DNA metabolic process 6.63E-05 rRNA processing 1.49E-06 histone H2A acetylation 1.96E-05 calcium-dependent cell-cell adhesion via plasma membrane cell adhesion molecules 1.06E-03 histone H4 acetylation 4.73E-04 cell-cell adhesion via plasmamembrane adhesion molecules 1.94E-03 regulation of protein phosphorylation 2.81E-03 cell junction assembly microtubule bundle formation C Direction translation 2.2103E-05 −1 Y537S allele specific Up n = 1644 Down n = 1496 Molecular and Cellular Functions [Y537S Genes] 2.75E-11 2 0 p-value regulation of transcription, DNAtemplated 3 1 Direction 2.59E-09 Z-score Mutant-specific shared Up n = 2241 Down n = 2303 Molecular and Cellular Functions [D538G Genes] mitotic cell cycle phase transition MSigDB Hallmark 2020 [Shared Mutant Genes] Direction p-value MSigDB Hallmark 2020 [D538G Genes] Direction p-value MSigDB Hallmark 2020 [Y537S Genes] 3.66E-03 Direction p-value E2F Targets 1.83E-18 E2F Targets 8.77E-13 Oxidative Phosphorylation 2.19E-15 G2-M Checkpoint 4.46E-12 G2-M Checkpoint 1.54E-11 mTORC1 Signaling 3.84E-14 Estrogen Response Early 2.25E-07 Mitotic Spindle 4.49E-03 Hypoxia 8.91E-07 Estrogen Response Late 5.76E-07 Myc Targets V2 7.34E-03 Myc Targets V1 9.01E-05 mTORC1 Signaling 5.76E-07 Epithelial Mesenchymal Transition 9.10E-03 PI3K/AKT/mTOR Signaling 8.53E-04 Androgen Response 3.13E-06 Estrogen Response Early 3.84E-03 Epithelial Mesenchymal Transition 7.59E-03 p53 Pathway 7.59E-03 D Wildtype Y537S Mutant D538G Mutant Supplemental Figure 5. ER-Y537S and ER-D538G mutants drive proliferative gene expression signatures in vivo (related to Figure 5). (A) Heatmap displays relative expression levels of mutant-specific shared, ER-Y537S specific, and ER-D538G specific genes. (B) Enrichr analysis highlights the molecular and cellular functions enriched in genes up- and down-regulated by both ER-D538G and ER-Y537S mutations (left), specific to ER-D538G (middle), and specific to ER-Y537S (right). (C) Enrichr analysis shows MSigDB Hallmark cancer pathways enriched in genes up- and down-regulated by both ER-D538G and ER-Y537S mutations (left), specific to ER-D538G (middle), and specific to ER-Y537S (right). (D) H&E staining is shown for wildtype, ER-Y537S, and ER-D538G xenograft tumors grown in the absence of supplemental estrogen. The wildtype tumor had extensive necrosis in the center of the tumor, the ER-Y537S had mottled necrosis throughout the tumor, and the ER-D538G had relatively little necrosis. Scale bar represents 1000 µm. 

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LEARNING BY IMAGES Official Publication of the Instituto Israelita de Ensino e Pesquisa Albert Einstein ISSN: 1679-4508 | e-ISSN: 2317-6385 Quantitative analysis in COVID-19: report of an initial experience Análise quantitativa na COVID-19: relato de uma experiência inicial Fábio Augusto Ribeiro Dalprá1, Eduardo Kaiser Ururahy Nunes Fonseca1, Gilberto Szarf1, Rodrigo Caruso Chate1 1 Hospital Israelita Albert Einstein, São Paulo, SP, Brazil. DOI: 10.31744/einstein_journal/2020AI5842 A B Figure 1. Chest computed tomography and superimposed 3DSlicer software quantification images. The upper series (A) show the findings when the patient returned to the emergency room, and the lower series (B) show the findings at the time of his clinical worsening. Axial sections of the chest tomography showing multifocal pulmonary ground-glass opacities predominantly peripheral and basal, more extensive in the last study, and quantitative images generated by the 3DSlicer software superimposed over the tomographic images. The areas marked in yellow show ground-glass opacities, those marked in green are areas of normal parenchyma, and those marked in orange are areas of consolidation. The extensive progression of the findings illustrates the numerical data provided How to cite this article: Dalprá FA, Fonseca EK, Szarf G, Chate RC. Quantitative analysis in COVID-19: report of an initial experience. einstein (São Paulo). 2020;18:eAI5842. http://dx.doi.org/10.31744/ einstein_journal/2020AI5842 Corresponding author: Eduardo Kaiser Ururahy Nunes Fonseca Avenida Albert Einstein, 627/701 – Morumbi Zip code: 05652-900 – São Paulo, SP, Brazil Phone: (55 11) 2151-1233 E-mail: eduardo.ururahy@einstein.br Received on: May 10, 2020 Accepted on: July 27, 2020 Copyright 2020 This content is licensed under a Creative Commons Attribution 4.0 International License. A 63-year-old man came to the emergency room complaining of unverified fever and myalgia. Oropharyngeal material was collected for reverse transcription testing followed by polymerase chain reaction (RT-PCR) for coronavirus disease 2019 (COVID-19), and a chest radiography was performed (normal), and the patient was discharged to home isolation, due to his mild symptoms, until the test result. After 3 days, the patient evolved with dyspnea, a drop in oxygen saturation (95%), and measured fever (38.6°C), and a chest computed tomography was performed (Figure 1) and he was admitted to hospital. The RT-PCR test for COVID-19 was positive. Three days later, his respiratory pattern worsened, with a decrease in oxygen saturation to 90%, and he was referred to a stepdown unit and a new tomography was performed (Figure 1). An 80-row CT scanner (Aquillion Prime, Canon Medical Systems, Tochigi, Japan) was used, with the patient in supine position, during maximum inspiration, and without injection of contrast medium. The following parameters were 1 einstein (São Paulo). 2020;18:1-2 Dalprá FA, Fonseca EK, Szarf G, Chate RC well-established, having even been used in the evaluation of pulmonary nodules in chest imaging.(5-7) Much has been studied since the beginning of the pandemic about the role of imaging tests in the prognosis and progressive control of COVID-19 patients, but a forceful answer has yet to be found. Our service, for instance, has been using the assessment of the tomographic progression of the disease as an auxiliary criterion in the clinical decision of hospitalization. The present case demonstrates the use of the 3DSlicer tool for the quantification of pulmonary tomographic changes, applied in the clinical monitoring of the patient, enabling an objective estimation of the involvement percentage and the progression rate of the disease. We believe that this tool can be an important resource for borderline cases or those that raise doubts about the significance of the progression. In addition, its association with artificial intelligence strategies can optimize the quantification process, rendering it possible the use of this quantification in a greater number of cases. used: reconstructions with slice-thickness of 1 mm, a tube voltage of 80 kVp to 120 kVp, and adjustable current, varying between 10 mA and 440 mA. The images of these two exams were then processed, using the 3DSlicer software to segment the normal parenchyma, groundglass opacities and consolidation areas in both lungs in the two exams performed for the patient (Figure 1). From this, a quantitative analysis was conducted, showing in the first study a total lung volume of 4,289.62cm³, with the right lung measuring 2,214.91cm³ and the left lung measuring 2,074.71cm³. The preserved parenchyma area measured 2,110.48cm³ (95.29%) in the right lung, and 2,056.79cm³ (99.14%) in the left lung. The right lung presented with 81.01cm³ (3.66%) of ground-glass opacities and 23.42cm³ (1.06%) of consolidations; the left lung presented with 14.98cm³ (0.72%) of ground-glass opacities, and 2.95cm³ (0.14%) of consolidations. In total, the patient had 2.85% of parenchyma affected by ground-glass opacities or consolidations in the first study. In the second study, the total lung volume calculated was 3,569.85cm³, with 1,814.95cm³ in the right lung and 1,754.90cm³ in the left lung. The preserved parenchyma area measured 915.17cm³ (50.42%) in the right lung, and 1,301.17cm³ (74.15%) in the left lung. The ground-glass opacity areas totaled 857.49cm³ (47.25%) in the right lung, and 447.63cm³ (25.51%) in the left lung, and the consolidation areas totaled 42.29cm³ (2.33%) in the right lung, and 6.09cm³ (0.35%) in the left lung. In three days of progression, the patient showed an increase of 1,259.93% in ground-glass opacity volume, with 958.47% in the right lung, and 2,888.06% in the left lung, and an 83.44% increase in the consolidation volume, with 80.57% in the right lung, and 106.66% in the left lung. These numbers resulted in a 46.81% reduction in the preserved parenchyma volume, with a reduction of 56.64% in the right lung, and 36.74% in the left lung. ❚❚AUTHORS’ INFORMATION Dalprá FA: http://orcid.org/0000-0003-2448-5826 Fonseca EK: http://orcid.org/0000-0002-0233-0041 Szarf G: http://orcid.org/0000-0002-1941-7899 Chate RC: http://orcid.org/0000-0002-4193-7647 ❚❚REFERENCES 1. World Health Organization (WHO). Emergencies preparedness, response. Novel Coronavirus – Republic of Korea (ex-Chin) [Internet]. Geneva: WHO; 2020 [cited 2020 Mar 30]. Available from: https://www.who.int/csr/don/21january-2020-novel-coronavirus-republic-of-korea-ex-china/en/ 2. World Health Organization (WHO). WHO Director-General´s opening remarks at the media briefing on COVID-19 -11 March 2020 [Internet]. Geneva: WHO; 2020 [cited 2020 May 12]. Available from: https://www.who.int/dg/ speeches/detail/who-director-general-s-opening-remarks-at-the-mediabriefing-on-covid-19---7-september-2020 3. Wu Z, McGoogan JM. Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: summary of a report of 72 314 cases from the Chinese Center for Disease Control and Prevention. JAMA. 2020;323(13):1239-42. 4. National Health Commission of the People’s Republic of China. New coronavirus cases rise to 571 in Chinese mainland [Internet]. Beijing (CHN): National Health Commission of the People’s Republic of China; 2020 [cited 2020 Mar 16]. Available from: http://en.nhc.gov.cn/2020-01/23/c_76004.htm 5. 3DSlicer Main Page. Welcome to the 3D Slicer Wiki pages [Internet]. 3DSlicer; 2019 [cited 2020 Mar 30]. Available from: https://www.slicer.org/ wiki/Main_Page 6. Kikinis R, Pieper SD, Vosburgh KG. 3D Slicer: a platform for subject-specific image analysis, visualization, and clinical support. in: Jolesz FA, editor. Intraoperative Imaging and Image-Guided Therapy. Springer: New York; 2014. p. 277-89. 7. Yip SS, Parmar C, Blezek D, Estepar RS, Pieper S, Kim J, et al. Application of the 3D slicer chest imaging platform segmentation algorithm for large lung nodule delineation. PLoS One. 2017;12(6):e0178944. ❚❚DISCUSSION Several cases of pneumonia of unknown origin that occurred in Wuhan, China, in late 2019, led to the discovery of a new type of coronavirus (2019-nCoV), called novel coronavirus-infected pneumonia (COVID-19).(1-4) The virus quickly spread and started to affect individuals outside the initial contagion area, in other countries and, finally, on all continents, and was declared a pandemic by the World Health Organization (WHO).(1-4) The 3DSlicer software is a free tool available online for download, whose use in quantitative imaging is einstein (São Paulo). 2020;18:1-2 2 

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Gobernanza de los Servicios Sociales municipales: dar voz y salida a las aspiraciones ciudadanas Governance of Municipal Social Services: Making Citizens Aspirations Known Enrique Pastor Seller Universidad de Murcia epastor@um.es Recibido: 8/10/2011 Revisado: 14/11/2011 Aceptado: 12/12/2011 Disponible on line: 15/02/2012 Resumen El artículo presenta un análisis de los mecanismos, prácticas y procesos de participación en los Servicios Sociales municipales y las dimensiones de análisis e intervención profesional con las que intensificar su influencia en el sistema. Se estructura en tres partes; la primera, alude a un breve marco teórico y normativo, enfatizando el análisis que las nuevas Leyes de Servicios Sociales autonómicas realizan respecto a la participación ciudadana y de las personas usuarias en el nivel local. La segunda presenta una caracterización, a modo de tipologías, de los mecanismos y procesos de participación en los Servicios Sociales municipales; y, por último, se sistematizan las propuestas y alternativas para mejorar el sistema y los procesos y prácticas orientados a intensificar la participación en la construcción de las políticas sociales en el nivel local. Palabras clave: Servicios Sociales, Administración Local, participación, políticas sociales municipales. Abstract The article presents an analysis of the mechanisms, practices and processes for participation in municipal social services and the dimensions of analysis and professional intervention with which they intensify their influence on the system. It is structured in three parts: the first refers to a brief theoretical and normative framework, emphasizing the analysis that the new Autonomous Social Services Laws make of the participation of citizens and users at the local level. The second section presents a characterization of types of mechanisms and processes of participation in municipal social services and finally, proposals and alternatives to improve the system and the processes and practices designed to enhance participation in the construction of social policies at the local level are systematized. Keywords: Social services, Local government, Participation, Municipal social policies. Referencia normalizada: Pastor Seller, E. (2012): «Gobernanza de los Servicios Sociales municipales: dar voz y salida a las aspiraciones ciudadanas». Cuadernos de Trabajo Social, 25(1): 143-158. Sumario: Presentación. 1. Breve descripción de la ruta metodológica. 2. Análisis de los niveles y expectativas de los procesos participativos. 3. Mecanismos y prácticas en el ámbito local: de lo institucional a las performances. 4. Análisis de la participación en el Sistema Autonómico de Servicios Sociales. 5. Caracterización y tendencias de los órganos de participación en Servicios Sociales. 6. Conclusiones y aportaciones desde los participantes para ejercer una ciudadanía activa. 7. Referencias bibliográficas. ción de los Servicios Sociales en el ámbito municipal de la región de Murcia, comparando sus resultados con realidades y tendencias observadas en otros estudios y experiencias de ámbito nacional e internacional. Se estructura Presentación El artículo presenta un análisis y evaluación de los mecanismos institucionalizados de organización de la participación ciudadana en el diseño, implementación, seguimiento y evaluaCuadernos de Trabajo Social Vol. 25-1 (2012) 143-158 143 ISSN: 0214-0314 http://dx.doi.org/10.5209/rev_CUTS.2012.v25.n1.38441 Enrique Pastor Seller en tres partes: la primera, alude a un breve marco teórico y normativo, enfatizando el análisis que las nuevas leyes de Servicios Sociales autonómicas realizan respecto a la participación ciudadana y a las personas usuarias del sistema en el nivel local. La segunda se centra en el análisis y evaluación de las oportunidades, contribuciones y limitaciones que presentan los órganos de participación institucionalizada en materia de Servicios Sociales municipales, a partir de las dimensiones objeto de investigación. La última sistematiza las propuestas y alternativas para mejorar el sistema en el ámbito municipal y, en segundo lugar, los procesos y prácticas que favorecerían intensificar la participación en la construcción de las políticas de Servicios Sociales en el nivel local. Centrar la investigación en la participación ciudadana en el ámbito de las políticas de bienestar social en general, y de los Servicios Sociales en particular en el ámbito municipal y a través de instrumentos participativos concretos y regulados, como son los Consejos locales, permite profundizar en un fenómeno emergente y complejo; adaptarse a la literatura especializada en torno a la democracia asociativa; disponer de un marco institucionalizado y una lógica normativa concreta y consolidada a la que referirse y establecer análisis comparativos entre contextos, mecanismos, prácticas y casos. Por otra parte, investigar acerca del impacto de los mecanismos de participación en la construcción de la agenda y su incidencia en la gestión de las políticas públicas, supone responder a un asunto innovador de interés preferente en el ámbito de las organizaciones sociales, administraciones públicas y entornos académicos, investigadores y profesionales. La participación, como señalan diferentes autores (Held, 2001; Warren, 2001; Löffler, 2004; Morales, 2005; Montero, Font y Torcal, 2006; Roque y Zimerman, 2009; Jorba y Anduiza, 2009; Pastor, 2011ª, entre otros), aporta sustantivos beneficios a la dinámica organizacional y comunitaria, al proporcionar una pro- Gobernanza de los Servicios Sociales municipales... gresiva adecuación del funcionamiento de las instituciones; romper la apatía y desconfianza ciudadana; ofrecer a los representantes herramientas para evaluar y mejorar la gestión de los asuntos públicos; permitir a la ciudadanía reconquistar y recuperar el espacio público; generar capital social; potenciar sentimientos comunitarios; permitir que la «política se socialice» y reforzar las decisiones a adoptar o, incluso, adoptadas. Por otra parte, las administraciones y organizaciones se ven sometidas a crecientes presiones para mejorar su calidad, eficacia y eficiencia, lo que hace necesario integrar, compartir y utilizar el conocimiento de sus clientes. De ahí que la participación se haya convertido en un asunto de interés preferente y transversal en la agenda de los gobiernos y profesionales que desean implementar una gestión política y técnica eficaz orientada a mejorar el bienestar social y la calidad de vida de la ciudadanía y de los usuarios de centros o servicios. La participación en la elaboración de las políticas sociales es: «una herramienta fundamental para mejorar el diseño, evaluar los servicios y detectar las necesidades reales de la población» (Subirats, 2007, p. 57). Pero esta centralidad requiere instrumentos que permitan una adecuada y transparente canalización de la misma y procesos de decisión y gestión descentralizada, colegiada, ágil y corresponsable en los asuntos públicos sociales municipales. El nivel local es un ámbito experimental para probar nuevos procedimientos de cooperación, así como formas innovadoras de articular liderazgo político y participación social (inteligencia cooperativa): governance1 local. Proporciona un ámbito privilegiado para revitalizar la democracia y es donde encontramos los primeros y mejores ejemplos de las nuevas formas de entender la gobernación de los asuntos públicos desde el modelo de municipio relacional. Pero los gobiernos locales, por una parte, ya no dialogan con una comunidad homogénea sino con múltiples comunidades e identidades que conviven y se interrelacionan en el territo- 1 La noción de gobernanza proporciona una nueva perspectiva para analizar la complejidad del proceso de toma de decisiones, generado por la pluralidad de actores involucrados que interactúan para formular, promover y lograr objetivos comunes, por medio del intercambio mutuo de conocimientos, recursos, ideas y normas. Lo nuevo es la complejidad del proceso y la variedad de actores del Estado, del mercado y la sociedad civil que participan con intereses divergentes en el proceso decisorio (Zurbriggen, 2011). 144 Cuadernos de Trabajo Social Vol. 25-1 (2012) 143-158 Enrique Pastor Seller rio, desde y con el mismo; por otra, la ciudadanía exige calidad y eficacia en la prestación de los servicios públicos, pero también participar en la misma definición y articulación de las políticas públicas que orientan el desarrollo social local. Ello supone tender hacia un gobierno local caracterizado y legitimado por lo relacional, por la capacidad de crear e impulsar redes, por estimular la participación de la sociedad civil y ejercer su liderazgo desde un nuevo modelo de gestión municipal más relacional y abierto y, por tanto, basado en la profundización democrática en clave más ciudadana y, por lo cual, de participación directa. Este modelo de «hacer política» y gestionar los asuntos públicos y la proximidad local favorecen construir pertenencias comunitarias, sentirnos implicados en los conflictos, en las necesidades y en la convivencia común y, por tanto, en las alternativas y decisiones finalmente adoptadas. La mayor parte de los gobiernos locales europeos se hallan, como señalan diferentes investigaciones y autores (Löffler, 2004; Colino y Del Pino, 2008; Ganuza y Frances, 2008, Navarro, Cuesta y Font, 2009, Pastor 2011b, entre otros), desde hace al menos dos decenios, inmersos en procesos de reforma. Los objetivos que se han perseguido con estas reformas pueden sintetizarse en dos: por una parte, las administrativas orientadas a lograr la eficiencia, la eficacia y la calidad de las estructuras administrativas locales y sus resultados en su relación con los deseos ciudadanos; y, por otra, las políticas pretenden lograr el enriquecimiento de la democracia local, normalmente en forma de mayor inclusióny acceso de la ciudadanía a las decisiones públicas que les afectan. 1. Breve descripción de la ruta metodológica El estudio, cuyos resultados se presentan, ha sido realizado en la Universidad de Murcia, entre 2004-2011, y ha permitido: a) disponer de una evaluación de las formas de organización de la participación ciudadana en los servicios de Bienestar Social de cada una de las corporaciones locales de la región; b) elaborar tipologías y análisis comparativos de casos; y c) evaluar el impacto, en términos de contribuciones y potencialidades de los Consejos, en la profundización democrática de las políticas públicas locales en materia de bienestar social, Cuadernos de Trabajo Social Vol. 25-1 (2012) 143-158 Gobernanza de los Servicios Sociales municipales... y la mejora en la eficacia de la gestión de los servicios y prestaciones sociales. Los contextos de investigación han sido todas las corporaciones locales (ayuntamientos y mancomunidades), con competencias en materia de Servicios de Bienestar Social de la región de Murcia, siendo las unidades de observación los mecanismos de participación implementados en éstos, con especial énfasis en los consejos e institutos de carácter general, territorial. En primer lugar, se identificaron los centros de Servicios Sociales de la región, los ámbitos de actuación y a los actores relevantes. Posteriormente, se realizaron entrevistas estructuradas a los responsables técnicos del universo de ayuntamientos y mancomunidades, orientadas a disponer de una tipología y caracterización del universo de mecanismos de participación existentes en cada municipio, comarca y en la región, concretamente (denominación, objeto, carácter, ámbito de actuación, evolución, grado de formalización, objetivos, composición, órganos y funciones). A continuación, se mantuvieron entrevistas en profundidad con los actores participantes en dichos mecanismos de participación, representando al universo de entidades (asociaciones, federaciones); universo de partidos políticos con representación política y técnicos vinculados a mecanismos de participación institucionalizada. Los actores debidamente seleccionados, atendiendo a la composición de los mecanismos de participación (políticos, profesionales, organizaciones sociales y vecinales) fueron entrevistados en varias ocasiones. El informe final, obtenido del proceso de análisis, se presentó a dos grupos de discusión, formados por actores cualificados de ámbito local y regional (políticos, profesionales y organizaciones sociales) y distintos de los entrevistados, para su análisis y sistematización de alternativas. Este proceso de investigación y análisis ha permitido disponer de una información validada, fiable y contrastable. 2. Análisis de los niveles y expectativas de los procesos participativos La ciudadanía «reclama una nueva forma» de gobernar y de relación e influencia reciproca y continua entre administración local y ciudadanía. Una profundización democrática, entendida como ampliación en términos cuantitativos y cualitativos, tanto de los actores (número y pluralidad representativa) que pueden interve145 Enrique Pastor Seller nir en los procesos participativos, como de las cuestiones y los niveles de influencia y producción de políticas sociales por parte de los diferentes actores implicados (políticos, organizaciones, técnicos y ciudadanos). De ahí, el indiscutible crecimiento de las experiencias participativas y de las políticas orientadas a impulsar la participación de la ciudadanía en los asuntos públicos, especialmente en el ámbito local. Una progresiva y evidente renovación y adaptación de las estructuras democráticas que, por medio de la participación e implicación más directa de la ciudadanía, contribuye a mejorar la democracia, fomentar una mayor transparencia, legitimidad, eficacia, eficiencia e influencia en las decisiones públicas; optimizar el rendimiento institucional y formar a mejores ciudadanos y políticos. Pero esta progresiva oferta de oportunidades de participación en las políticas públicas convive con contextos políticos e institucionales reticentes al impulso de políticas orientadas a promover la participación y la apertura de nuevos espacios y procesos participativos y que prefieren circunscribirse a mecanismos tradicionales de la democracia representativa y delegativa. La participación ciudadana puede contener diferentes significaciones, atendiendo a los múltiples objetivos a los que puede dirigirse un proceso participativo y, en razón de éstos, diseñar los mecanismos e indicadores que pueden facilitarlo y evaluarlo, respectivamente. La clasificación de los objetivos, del porqué de la participación, puede realizarse en función de diferentes criterios, destacando dos: el papel otorgado a la ciudadanía y los resultados que los gobernantes/responsables esperan obtener. Respecto al papel otorgado a la ciudadanía podemos distinguir: — Información y formación. Es habitual confundir participación con dar información. Sin embargo, ésta es sólo el primer nivel o requisito previo (perspectiva del continuum), dado que disponer de información es imprescindible para que pueda existir participación, pero no es suficiente. La información circula en dos sentidos: ascendente y descendente. Lo importante no es solo la cantidad de información, sino el momento (antes o después de haber adoptado las decisiones) y su calidad (relevancia para sus intereses y forma de ofrecerla). 146 Gobernanza de los Servicios Sociales municipales... — Consulta. Implica que la población no sólo conoce propuestas y decisiones, sino que tiene opciones de expresar opiniones, sugerencias y alternativas y será eficaz cuando la ciudadanía disponga de evidencias de que se le ha escuchado de manera concreta. Se produce cuando las autoridades o responsables deciden mejorar sus decisiones, teniendo en cuenta la opinión de determinados grupos o individuos afectados (usuarios/as) o del público en general. Requiere que los temas planteados se encuentren en proceso de diseño o puedan ser potencialmente modificados, lo que implica una clara voluntad política y técnica de incorporar dichas opiniones y la capacidad institucional y normativa para introducir dichos cambios. El nivel de consulta deliberativa más alto es aquel que se basa en la interacción constructivista o transacción, lo que supone intercambios circulares en los que la persona, grupo y entorno se moldean recíprocamente en continuos e indeterminados procesos de influencia mutua. La consulta puede ser preceptiva o discrecional. — Delegación y cogestión. Supone el tercer peldaño en la escalera e implica la intervención ciudadana, de los/as usuarios/as y representantes en el proceso de elaboración de las decisiones, mediante estructuras establecidas, bien territoriales, sectoriales u organizacionales. Los participantes o usuarios, mediante acciones directas o a través de organizaciones, hacen efectivo su poder y capacidad de escoger una o varias alternativas en función de sus propios intereses y necesidades. Un nivel que tiende hacia formas de concertación, control de la administración, cogestión y autogestión ciudadana, al darse una participación directa en la gestión de políticas y/o en el diseño de las mismas. En cuanto a lo esperan obtener los gobernantes, podemos distinguir dos tipos de objetivos: los de legitimidad y mejora de las decisiones y otros de nueva generación, tales como disponer de colaboradores y generar capital social. 3. Mecanismos y prácticas en el ámbito local: de lo institucional a las performances El complejo, heterogéneo y dinámico escenario de mecanismos y prácticas participativas existentes en el ámbito municipal suelen distinguirse en relación con los mecanismos de base asociativa (consejos municipales de participación ciuCuadernos de Trabajo Social Vol. 25-1 (2012) 143-158 Enrique Pastor Seller dadana; consejos territoriales o sectoriales; comités de usuarios de los servicios y agrupaciones de desarrollo local; los procesos y prácticas directas o deliberativas (presupuestos participativos; jurados ciudadanos o núcleos de intervención participativa; asambleas de ciudadanos; reuniones vecinales, comunales o de servicios; referenda o consultas populares; manifestaciones, huelgas, boicots, encuestas de satisfacción; encuestas deliberativas; grupos de discusión, etc.) y los mecanismos y prácticas mixtos (planes estratégicos territoriales; agendas21; consejos, foros o asambleas territoriales, sectoriales o de servicios; plataformas ciudadanas; talleres de prospectiva de territorios y servicios, etc.). Del análisis de las investigaciones sobre participación en el ámbito local (Alguacil, 2008; Brugué y Vallés, 2005; Colino y Del Pino, 2008; F.E.M.P. 2002; Font, 2001; Gutiérrez, 2005; INAP, 2008; Navarro, 2008; Navarro, Cuesta y Font, 2009; Montero, Font y Torcal, 2006; Morales, 2005; Pastor 2011b; Pindado, 2008; Rodríguez et.al., 2005; Rodríguez y Ajangiz, 2007; Ruiz, 2006; Subirats, 2007; Pastor, 2009a, y otros autores), podemos constatar dos fenómenos: por una parte, la descentralización del Estado de bienestar, a partir de mediados de los años ochenta, que ha provocado que los gobiernos municipales hayan tenido que buscar interlocutores sociales —tercer sector o sistema e incluso mercantiles— en relación con sus nuevas competencias; y, por la otra, que desarrollen estrategias adaptativas en relación con las rasgos estables y dinámicos de su estructura política en su trayectoria histórica. De ahí, que la oferta de oportunidades de participación política que impulsan, inicialmente, los ayuntamientos fundamentalmente urbanos (Navarro, 2008), se basen en el modelo tradicional y típicamente asociativo (consejos territoriales y sectoriales), aunque se van poniendo en marcha cada vez más mecanismos de participación directa —modelo de orientación «ciudadanista»— bien de información o consulta (encuestas, defensor del ciudadano, referéndum e internet) o de deliberación, que suponen un proceso de discusión pública en torno a decisiones o iniciativas concretas (presupuestos participativos y consejos ciudadanos). 2 3 Gobernanza de los Servicios Sociales municipales... En la actualidad, observamos con nitidez acciones colectivas de protesta mediante dramatización de la contienda política —Salimos a la calle a protestar— a través de diferentes formas o performances (huelgas, encadenamientos, caceroladas, concentraciones ciudadanas, motines, abrazos y desnudos colectivos, manifestaciones, toma de edificios, impedimentos a desahucios, marchas, piquetes, etc.) orientados a sensibilizar y ejercer influencia política, social y económica. En este sentido, Tilly y Tarrow (2006) señalan que las performances son las formas, relativamente familiares y estandarizadas, con las que un conjunto de actores profieren una serie de gritos colectivos a conjunto de actores políticos. Según Tilly (2008), estas son en parte el resultado de procesos de innovación y aprendizaje. Los actores van registrando la eficacia y la adecuación de las acciones de protesta que desarrollan, adaptándolas, mejorando los recursos y las iniciativas necesarias para su desarrollo, la división de tareas, la necesidad de implicar a otros, etc.; y, si se quiere, aprendiendo cómo resolver los dilemas de movilización y coordinación que supone la acción colectiva que pretenden desarrollar, hasta asignarles una denominación compartida que, en su repetición es reconocida por otros (Herrera, 2010) 4. Análisis de la participación en el Sistema Autonómico de Servicios Sociales El conjunto de leyes autonómicas de Servicios Sociales contemplan, aunque a diferentes niveles, el principio de «participación ciudadana» o «cívica», mediante la creación de cauces y mecanismos con los que impulsar la implicación de la ciudadanía en la gestión del sistema público de Servicios Sociales, así como en la planificación, seguimiento, control y evaluación de los centros, planes y programas sociales. 4.1. La participación como finalidad y principio Las leyes más recientes identifican la promoción de la participación como finalidad y principio rector del sistema (Ley de Cantabria 2/20072, Ley 5/2009 de Aragón3, Ley 12/2008 Ley de Cantabria 2/2007, de 27 de marzo de Derechos y Servicios Sociales (BOC núm. 66, de 3 de abril). Ley 5/2009, de 30 de junio, de Servicios Sociales de Aragón (BOA núm. 132, de 10 de julio). Cuadernos de Trabajo Social Vol. 25-1 (2012) 143-158 147 Enrique Pastor Seller del País Vasco4, Ley 14/2010 de Castilla-La Mancha5, Ley 16/2010 de Castilla y León6); como objetivo (Ley Foral 15/20067; Ley 12/2007 de Cataluña8; Ley 13/2008 de Galicia9; Ley 5/2009 de Aragón; Ley 4/2009 de las Illes Baleares10; Ley 7/2009,de La Rioja11; Ley 14/2010 de Castilla-La Mancha); como prestación (Ley del Principado de Asturias 1/200312, Ley 14/2010 de Castilla-La Mancha) de las políticas de Servicios Sociales, como competencia municipal y función específica de los Servicios Sociales de base. Atendiendo a las últimas leyes promulgadas, la Ley 14/2010 de Castilla-La Mancha, reconoce la participación como objetivo13, principio rector14 y prestación técnica15 de los Servicios Sociales de Atención Primaria, mediante la potenciación de la implicación y la responsabilidad social, con el fin de conseguir que las personas y grupos sean agentes activos en la generación de alternativas de mejora, colaborando con los Servicios Sociales. Se considera una prestación transversal, ya que a través de ella se promueven actitudes favorecedoras de la convivencia ciudadana, afianzando el entorno comunitario como contexto que garantice la continuidad de los cambios conseguidos (art. 36 h). De igual forma, la Ley 16/2010 de Castilla y León contempla la participación como finalidad y principio que ha de regir el sistema y por tanto que ha de ser fomentada, facilitada y garantizada por los poderes públicos a todos los niveles: ciudadanos, Gobernanza de los Servicios Sociales municipales... entidades de iniciativa social, agentes sociales y personas usuarias. 4.2. Mapa de los mecanismos de canalización de la participación asociativa y directa Contemplan la creación de mecanismos que encaucen la participación ciudadana, así como los derechos y deberes de las personas usuarias de centros, servicios y programas, bien directamente de manera individual o a través de entidades sociales representativas, (no lucrativas, iniciativa social y voluntariado). Ambos ejercicios de ciudadanía se plasman especialmente, en las carteras de servicios de las leyes promulgadas a partir de 2005, que refuerzan la protección de las personas usuarias, con un principio de garantía de la participación y una descripción detallada de derechos y deberes, entre los que se identifica la participación de las personas como agentes de su propio cambio y de los grupos y entidades de la sociedad civil en el funcionamiento del sistema de Servicios Sociales. La participación ya estaba presente en las primeras leyes de Servicios Sociales de los años 1980 y 1990 (y en su desarrollo normativo posterior), la novedad radica en su extensión a las carteras o catálogos de servicios, su vinculación a la calidad y a los derechos y deberes de las personas usuarias y profesionales, y al fortalecimiento de la iniciativa social en la provisión de servicios y libre elección del consumidor en la creciente externalización de los mismos. 4 Ley 12/2008, de 5 de diciembre, de Servicios Sociales del País Vasco (BOPV núm. 246, de 24 de diciembre). 5 Ley 14/2010, de 16 de diciembre, de Servicios Sociales de Castilla-La Mancha (BOE núm. 38, de 14 de febrero de 2011). 6 Ley 16/2010, de 20 de diciembre, de Servicios Sociales de Castilla y León (BOE núm. 7, de 8 de enero de 2011. BOCYL. núm. 244, de 21 de diciembre de 2010 y corrección de errores en BOCYL, núm. 23, de 3 de febrero de 2011). 7 Ley Foral 15/2006, de 14 de diciembre, de Servicios Sociales ( BOE núm. 27, de 31 de enero). 8 Ley 12/2007, de 11 de octubre de Cataluña (BOE núm. 266, de 6 de noviembre). 9 Ley 13/2008, de 3 de diciembre, de Servicios Sociales de Galicia (DOG núm. 245, de 18 de diciembre). 10 Ley 4/2009, de 11 de junio, de Servicios Sociales de las Illes Baleares (BOBB núm. 89, de 18 de junio). 11 Ley 7/2009, de 22 de diciembre, de Servicios Sociales de La Rioja (BOR núm. 166, de 28 de diciembre). 12 Ley del Principado de Asturias 1/2003, de 24 de febrero, de Servicios Sociales (BOPA de 8 de marzo). 13 El sistema tiene entre sus objetivos: «crear y reforzar cauces de participación de la comunidad en la resolución de las necesidades sociales y en particular la participación individual y organizada de las propias personas usuarias y de las entidades activas en el ámbito de los Servicios Sociales» (art. 5 h). 14 Entendida como: «la participación de la ciudadanía, individual o colectivamente, así como la de entidades públicas y privadas en el diseño y desarrollo del Sistema, será garantía de eficacia del mismo» (art. 6 i). 15 Incluida en el catálogo de prestaciones técnicas de Servicios Sociales de Atención Primaria. 148 Cuadernos de Trabajo Social Vol. 25-1 (2012) 143-158 Enrique Pastor Seller Con el fin de garantizar la participación en la planificación y gestión del sistema público de Servicios Sociales, contemplan planes estratégicos y sectoriales, así como la creación de órganos asesores y consultivos de participación directa y asociativa, tales como: a) Mecanismos regionales de coordinación interadministrativa e interdepartamental para elaborar la planificación autonómica (planes estratégicos, mapas) y garantizar la participación de todas las administraciones competentes. Contemplan órganos colegiados tales como: observatorios y consejos de coordinación interinstitucional o interadministrativa. b) Comités técnicos de estudio de necesidades sociales y evaluación de la eficiencia y calidad de los Servicios Sociales y comités de ética de los Servicios Sociales16. c) Órganos regionales de participación, asesoramiento, consulta y propuesta en materia de Servicios Sociales, adscritos a la consejería competente, cuyo objeto es articular la participación y contribuir al mejor desarrollo, calidad y eficacia del sistema (consejos autonómicos de acción social, Servicios Sociales, mesas de Servicios Sociales, etc.). d) Órganos regionales asesores y consultivos en asuntos relativos a la atención a la dependencia, adscritos a la Consejería competente en la materia (Comité Consultivo de Atención a la Dependencia). e) Órganos colegiados sectoriales, creados al efecto por sectores de la acción social. f) Órganos provinciales, forales o insulares de participación, asesoramiento, consulta y propuesta en ese ámbito en materia de Servicios Sociales, adscritos a la Diputación Provincial, Foral o Insular (consejos de Servicios Sociales). Gobernanza de los Servicios Sociales municipales... g) Mecanismos locales de participación en Servicios Sociales (consejos locales de Servicios Sociales). h) Fomento de la participación de la iniciativa social en los Servicios Sociales. En este aspecto nos encontramos con leyes que diferencian la iniciativa social lucrativa (iniciativa mercantil) de la no lucrativa (iniciativa social). En otras, dicha diferenciación no existe, reconociendo por igual al derecho de participar en la creación de centros y servicios y únicamente ante análogas condiciones de calidad, eficacia y costes, darán prioridad a la colaboración con entidades de iniciativa social sin ánimo de lucro17. En cuanto a las modalidades de colaboración se reconocen fórmulas tales como: concertación, convenios y acuerdos y subvenciones. i) Escasas leyes, y de manera ambigua, señalan que podrá articularse a través del movimiento asociativo y con procesos participativos que la Administración de la autonómica y las entidades locales competentes en materia de Servicios Sociales dispongan para canalizar la información, la propuesta, el debate o la consulta en relación con las singulares actuaciones de planificación, seguimiento y evaluación que les competan. 4.3. Participación, derechos y deberes de las personas usuarias Respecto a la participación de las personas usuarias, las leyes de «segunda» y «tercera» generación les atribuyen, al menos formalmente, un papel más activo, concretamente: participar en todas las decisiones que les afecten de forma directa o indirecta, individual o colectiva.Todas las entidades y centros de Servicios Sociales deberán contar con procedimientos de participación democrática de las personas 16 «El Comité de Ética (…) de las Illes Balears es un órgano colegiado consultivo, interdisciplinario e independiente. Sus finalidades son sensibilizar al personal (…) respecto de la dimensión ética (…) garantizar el derecho (…) dignidad humana e intimidad, sin ninguna discriminación, así como identificar, analizar y evaluar los aspectos éticos de la práctica social» (art. 46 Ley 4/2009 de Servicios Sociales de las Illes Balears 17 Ley 13/2008 de Galicia en su art. 33.3, así como la Ley 5/2009 de Aragón, señalan para el establecimiento de conciertos, que las Administraciones públicas darán prioridad, cuando existan análogas condiciones de eficacia, calidad y rentabilidad social, a las entidades prestadoras de servicios de naturaleza social o sin ánimo de lucro (art. 25). En igual sentido, la Ley 16/2010 de Castilla-León cuando aborda el fomento de la iniciativa social sin ánimo de lucro señala que «Aante análogas condiciones de calidad, eficacia y costes, darán prioridad a la colaboración con entidades de iniciativa social sin ánimo de lucro» (art. 87.2). Cuadernos de Trabajo Social Vol. 25-1 (2012) 143-158 149 Enrique Pastor Seller usuarias, o de sus representantes legales, de acuerdo con lo que se determine reglamentariamente; a tal efecto establecen como mecanismo el Consejo de usuarios. Resulta de interés detenernos en los derechos y deberes de las personas usuarias que incorporan las recientes leyes con una clara influencia por la Ley 39/200618, de Promoción de la Autonomía Personal y Atención a las personas en situación de Dependencia. Entre los derechos destacan: recibir información suficiente, veraz, accesible y comprensible sobre prestaciones, requisitos, derechos y deberes que correspondan; acceder y recibir atención en condiciones de igualdad, sin discriminación; realizar valoración y diagnóstico de situación y necesidades y participación de resultados; disponer de plan individual de atención social con participación del interesado, representante o familia en toma de decisiones; asignar un profesional de referencia que coordine Plan y dote de integralidad la intervención; dar consentimiento específico y libre y participar en toma de decisiones; proporcionar atención individualizada y urgente ante supuestos de emergencia; formular quejas y reclamaciones sobre atención y prestaciones; recibir servicios y prestaciones conforme a criterios de calidad establecidos y participar en planificación, seguimiento y evaluación de los Servicios Sociales. En cuanto a los deberes: comparecer y facilitar información veraz y completa para valoración o atención; cumplir normas, requisitos, procedimientos de acceso y disfrute y compromisos adquiridos; seguir el plan individual de atención social y orientaciones técnicas; destinar prestaciones a tal fin; mantener conductas de respeto, responsabilidad, tolerancia, convivencia y colaboración, cumpliendo con las obligaciones; respetar la dignidad y los derechos del personal; y utilizar instalaciones con responsabilidad. Gobernanza de los Servicios Sociales municipales... Como conclusión, podemos destacar la importancia concedida a la persona usuaria o beneficiaria respecto a la toma de decisiones en el proceso de diagnóstico e intervención social y su vinculación a criterios de calidad. 4.4. La participación como derecho y deber de los profesionales Las leyes promulgadas tras la Ley de Dependencia contemplan derechos y deberes de los profesionales de Servicios Sociales, concretamente: formarse de manera continúa y adecuada al contenido de la actividad que hayan de desarrollar y conocer las herramientas técnicas y tecnológicas que les sean precisas. Para ello, deberán contar con medios y apoyos necesarios para desarrollar su actividad con calidad, eficacia y eficiencia y las administraciones han de adoptar las medidas pertinentes para la prevención y atención de las situaciones de riesgo derivadas de su trabajo, garantizando su integridad. Se destaca en todas ellas el deber de dispensar a las personas usuarias de los Servicios Sociales, a los responsables de estos servicios y a los demás profesionales, un trato digno y correcto con respeto a su intimidad, y el derecho a ser tratado por todos ellos con respeto y corrección. Atendiendo al objeto del análisis de este artículo, cabe destacar que algunas leyes contemplan el derecho y el deber de formar parte de los órganos de participación y de intervenir en los procesos de evaluación de los servicios19. 4.5. Calidad y sostenibilidad La calidad en las leyes de «tercera generación» constituye un derecho de las personas usuarias y, en consecuencia, un principio20 y objetivo prioritario del sistema, regulando normativas específicas (como la Ley 3/2007, de 1 de marzo, de calidad de los Servicios Sociales21), organismos específicos (observatorios); criterios, estándares y procesos de calidad específicos 18 Ley 39/2006, de 14 de diciembre, de Promoción de la Autonomía Personal y Atención a las personas en situación de Dependencia (BOE núm. 299, de 15 de diciembre de 2006). 19 Tal y como recoge el art. 66 de la Ley 5/2009 de Aragón; art.56 b de la Ley 16/2010 de Castilla y León o en el art. 11.3 f de la Ley 14/2010 de Castilla-La Mancha. 20 Así, como ejemplo, la Ley 3/2008 de Galicia identifica como principio la gestión de los Servicios Sociales con criterios de «economía, eficacia y eficiencia», debiendo utilizar la Administración los instrumentos idóneos para cada intervención, de forma que los objetivos y estándares de calidad se consigan con independencia de la titularidad de la gestión, mediante un uso racional de recursos públicos (art. 4 n). 21 Publicada en el BOE núm. 76, de 29 de marzo de 2007. 150 Cuadernos de Trabajo Social Vol. 25-1 (2012) 143-158 Enrique Pastor Seller (estrategias, plan de calidad), e incluso alguna de ellas «premiando» la calidad. Así, como ejemplo, la Ley 14/2010 de Castilla-La Mancha regula que aquellas entidades que presten Servicios Sociales con niveles de calidad o excelencia superiores a los estándares mínimos que se regulen en el catálogo, puedan obtener la correspondiente acreditación que lo reconozca, la cual podrá ser considerada como mérito o mejora en los procedimientos de concurrencia pública que la Administración realice para la provisión de prestaciones del sistema público de Servicios Sociales. Por último, cabe destacar la creciente preocupación de las administraciones públicas por la sostenibilidad del sistema, evidenciada en su inclusión en las últimas leyes y concebida como financiación suficiente del sistema para asegurar su estabilidad y continuidad en el tiempo, lo que provoca, entre otros efectos: la expansión del mercado y del tercer sector por la vía de la prestación de servicios y la internalización del coste de la protección a la dependencia por la vía del copago y la atención familiar informal, característico del modelo social europeo de atención a la dependencia (Rodríguez Cabrero, 2011) y que ha tenido consecuencias en la configuración del sistema de Servicios Sociales (Casado, 2010, Vila, 2011). 5. Caracterización y tendencias de los órganos de participación en Servicios Sociales Los mecanismos de participación ciudadana mayoritarios en las políticas de Servicios Sociales municipales son los siguientes: a) estructuras de participación de base asociativa o consejos22; b) organismos públicos autónomos desconcentrados o institutos23 y c) mecanismos no formales de participación de base asociativa 24. Gobernanza de los Servicios Sociales municipales... Los resultados de la investigación, a partir de las dimensiones de análisis y desde los actores participantes en los mecanismos de participación en la región, permiten identificar potencialidades, limitaciones y tendencias de los consejos territoriales de Bienestar Social en su capacidad para influir en los procesos de democratización en la construcción de las políticas de Servicios Sociales en el ámbito municipal, siendo los más significativos los siguientes: — Existencia de un marco normativo propicio y proactivo para la creación, impulso y consolidación de órganos de gestión desconcentrada y participación ciudadana tanto general/territorial como por áreas de competencia municipal, sectores de población y/o problemáticas sociales. — Un modelo de participación institucional que otorga a la administración local, a través del responsable político y/o técnico del área, una enorme y flexible capacidad de control sobre estrategia, oportunidades, agenda, asuntos, actores participantes y procesos participativos. — La composición y representatividad de los consejos tienen una base de participación, fundamentalmente, asociativa; con un claro protagonismo de las federaciones, fundaciones y asociaciones frente a los ciudadanos de forma individual, plataformas y entidades minoritarias; a la vez que se privilegia la representación sectorial a la territorial. — Dificultades de representación y participación plural real ante la fragmentada y atomizada realidad asociativa, especialmente en los municipios de mayor tamaño donde el tejido social es muy numeroso y diverso. 22 Se caracterizan por ser consultivos, de ámbito territorial y/o sectorial, de carácter extensivo y facilitadoras de información, asesoramiento y captación de propuestas. 23 Disponen de personalidad jurídica propia, delegación de competencias en Servicios Sociales generales y sectoriales y con cierta autonomía en la toma de decisiones y la gestión económico-administrativa y técnica del área. De esta forma, combinan: adopción de decisiones, ejecución/gestión de acuerdos y participación. 24 Dimensión informal del ejercicio de facilitación. A través de reuniones periódicas protagonizadas por técnicos de Servicios Sociales con organizaciones del territorio y técnicos de otros sistemas. La iniciativa, convocatoria, agenda y organización se realiza a través de los directores o coordinadores de los centros de Servicios Sociales, en ocasiones sin respaldo en los asuntos tratados y acuerdos alcanzados en ellas. Entre sus fines: ofrecer información, detectar necesidades; solicitar participación, promover la cooperación interasociativa y generar redes y protocolos de colaboración técnica interinstitucional. Cuadernos de Trabajo Social Vol. 25-1 (2012) 143-158 151 Enrique Pastor Seller — Existencia de contextos asimétricos de poder entre las organizaciones sociales ante la desigual capacidad y oportunidad de acceder a espacios de decisión de los asuntos públicos por parte de estas. Se observa que ciertas organizaciones sociales acaparan la representación social en múltiples foros participativos. — La desigual capacidad y habilidad de los participantes en formular opiniones políticas en los mecanismos de participación caracterizados, en ocasiones, por lenguajes o documentos tecnocráticos o burocráticos provoca la exclusión de representantes y las «opiniones» consideradas «no cualificadas», intensifica la asimetría representativa de colectivos y problemáticas sujetos y objeto de la acción de los Servicios Sociales y, a la vez, produce sentimientos de «inhabilitación» a la acción política por parte de personas con escasas habilidades comunicativas y argumentación técnica. — Objetivos de carácter consultivo, no vinculantes para las autoridades (dar y recoger información) y, por lo tanto, basados en un concepto restringido de participación, entendida en términos de información, consulta y colaboración. — Existencia de una brecha entre objetivos formales (recogidos en reglamentos) y reales que se alcanzan en la práctica, así como en la distinta percepción que tienen los actores implicados acerca de los objetivos que deben o debieran cumplir (expectativas). — Favorable valoración de las organizaciones sociales respecto de los mecanismos de participación en razón de la posibilidad de mejorar el diálogo e intercambio de información con los profesionales de los Servicios Sociales. Por su parte, los técnicos lo valoran por ser un instrumento para detectar necesidades sociales, agilizar procesos y, en ocasiones, mejorar la coordinación, de las cuestiones cotidianas del área (seguimiento de casos, altas y bajas en prestaciones económicas y servicios, información de proyectos y resultados). — La comunicación impulsada a través de los mecanismos de participación ha permitido mejorar, en algunos casos, la derivación de casos y el desarrollo de acciones concretas entre los centros de Servicios Sociales y las organizaciones sociales. — Se constata una progresiva burocratización, profesionalización, especialización fun152 Gobernanza de los Servicios Sociales municipales... cional y la dependencia del sector asociativo respecto de las administraciones públicas. Los procedimientos y condiciones de acceso al sectorial, y cada vez más concurrido y «exiguo», «fondo» público (contratos, acuerdos, subvenciones), puede llevar a las entidades a reducir su potencial de flexibilidad y capacidad de innovación, dado que al final es la Administración quien determina qué (centros y servicios), para qué (sentido y fines), cómo se hace (modos de intervención) y a quién (colectivos y/o problemáticas diana) se han de prestar los servicios. — El dominio de la lógica pública, en un contexto relacional caracterizado por una elevada dependencia económica y de prestación de servicios, somete a las organizaciones a dilemas, sobre la propia identidad, autonomía, ética y las estrategias de intervención social, provocando relaciones interasociativas en las que predomina la competencia sobre la cooperación, las redes o las alianzas y que nos diferencian de modelos de bienestar más consolidados, como el nórdico o el bismarkiano, en los que estas entidades comparten responsabilidades con la Administración. — El proceso participativo es percibido y considerado un trámite más administrativo que sustantivo, más vinculado con el cumplimiento de plazos, reglamentos, etc., y en el que el análisis y la deliberación de problemáticas y cursos de acción política a implementar y evaluar quedan al margen o bien son «rescatados» para dar cumplimiento de requerimientos técnicos o administrativos y no de carácter político. — La gestión técnico-administrativa de los asuntos sociales adquiere carácter preferente en la agenda, organización, funcionamiento y dinámica de estos mecanismos. El proceso participativo es dirigido por el discurso técnico a partir de las cuestiones decididas políticamente o como consecuencia de ellas, desestimulando a la participación. — Los mecanismos de participación no son percibidos por las organizaciones como un espacio propio, sino como un punto de encuentro periódico con la Administración Local en el que ésta les ofrece información acerca de las actuaciones realizadas o a realizar en materia de política social. Sus participantes se convierten en «consumidores invitados» de la oferta de participación, pero no en protagonistas, Cuadernos de Trabajo Social Vol. 25-1 (2012) 143-158 Enrique Pastor Seller pueden hacer valer su voz pero filtrada en atención a su encaje en la agenda y estrategia de la acción política. — Desconocimiento ciudadano y de las entidades no participantes de la existencia de los Consejos o de su funcionamiento, motivado por un proceso de formalización y funcionamiento no acompañado de actuaciones previas y posteriores suficientes, de información, difusión, consulta, propuesta y debate. Esta insuficiente transparencia retroalimenta o contribuye a la arbitrariedad en la selección de actores y funcionamiento de los mecanismos, reproduciendo sesgos en la participación. En conclusión, el análisis de las políticas de participación ciudadana en los Servicios Sociales municipales revela que la mayoría de los ayuntamientos han puesto en marcha consejos; posibilitan espacios de participación en niveles de información y en ocasiones de consulta y reservan la toma de decisiones a los órganos del gobierno municipal. A los consejos los actores acuden con una información documentada y exhaustiva, pero también sesgada y filtrada por quién ofrece la información, el qué y el cómo de la misma, lo que determina la formación de las opiniones individuales y colectivas respecto a la realidad y las alternativas que se presentan como objetivas y posibles. La escasa vinculación de los acuerdos y la percepción de no influir en la política social local de manera práctica conlleva que los miembros se sientan, en ocasiones, «invitados», «no participantes» — tal y como lo manifiestan— en el proceso de toma de decisiones reales, apareciendo situaciones de absentismo o inasistencia a las sesiones. Las organizaciones «enmudecen», adoptan un papel «ausente» e incluso «conformista» en las sesiones, utilizando otras vías más útiles para canalizar sus demandas, como se advirtió anteriormente, y para influir en la toma de decisiones. 6. Conclusiones y aportaciones de los participantes para ejercer una ciudadanía activa En coincidencia con las conclusiones de estudios sobre la democracia asociativa (Löffler, 2004; Brugue, Font y Gomá, 2005; Montero, Font y Torcal, 2006; Colino y Del Pino, 2008; Navarro, 2008; Navarro, Cuesta y Font, 2009; Pastor, 2011b), la política municipal de particiCuadernos de Trabajo Social Vol. 25-1 (2012) 143-158 Gobernanza de los Servicios Sociales municipales... pación en los Servicios Sociales municipales conforma un modelo democrático que puede denominarse de «democracia de acceso», al quedar fuera la incorporación de la ciudadanía en la formulación efectiva de las políticas, no ya en la activación de la agenda, sino en la discusión en torno a los problemas que ésta debe o no debe incluir. Los consejos e institutos favorecen los procesos de interacción de los actores, pero no garantizan por sí mismos, y de forma automática, una democratización en la construcción de las políticas sociales en el ámbito local ni viceversa. No favorecen la posibilidad de realizar un ejercicio de poder efectivo, no ya en la activación de la agenda, sino en la discusión de los problemas que debe o no debe incluir. De ahí que más allá de su regulación, precisa un diseño, una organización y un funcionamiento que posibilite unas condiciones y oportunidades reales para acceder e influir en los procesos de toma de decisiones respecto de las políticas sociales. Atendiendo al análisis desde los participantes de la investigación, podemos sistematizar las propuestas orientas a mejorar: a) el sistema de Servicios Sociales en el ámbito municipal y b) los mecanismos y prácticas participativas. 6.1. Aportaciones para impulsar mejoras en el sistema de Servicios Sociales en el ámbito municipal 6.1.1. Mayor autonomía normativa, económica y de gestión de la administración local en materia de Servicios Sociales de atención primaria Transferir a las corporaciones locales el presupuesto y las competencias necesarias para una adecuada y autónoma gestión de los servicios y elaborar planes estratégicos en cada una de las corporaciones locales. 6.1.2. Implantar un sistema organizativo en la Administración Local que promueva las políticas públicas de manera participativa Impulsar la creación y consolidación de concejalías de «participación ciudadana» que promuevan órganos de participación asociativa y de participación directa de los ciudadanos en las diferentes áreas de gestión municipal; impulsar la elaboración de políticas sociales transversales desde la coordinación de las dife153 Enrique Pastor Seller rentes áreas de competencia municipal, atendiendo a la participación como elemento esencial para su configuración y alcanzar acuerdos políticos que eviten la instrumentalización partidista de los mecanismos. 6.1.3. Crear instrumentos normativos que regulen el derecho y los mecanismos participativos en la Administración Local Impulsar medidas que garanticen la participación de las personas y entidades sociales en los procesos de toma de decisiones en los asuntos sociales públicos: promover Reglamentos Municipales de Participación Ciudadana, elaborar y aprobar «cartas» de los derechos de los ciudadanos e incorporar las posibilidades de las nuevas tecnologías para innovar en la participación y crear registros municipales accesibles. 6.1.4. Universalizar la cobertura de consejos territoriales y sectoriales de Servicios Sociales en todas aquellas corporaciones locales con competencia en la gestión de los Servicios Sociales de Atención Primaria Crear y consolidar los consejos municipales de Servicios Sociales generales, generando estructuras intermedias de participación de carácter sectorial y territorial ampliando la cobertura de participantes y la participación directa de la ciudadanía y vinculando acuerdos y propuestas de manera ascendente y descendente. 6.1.5. Desconcentrar y descentralizar la gestión de los Servicios Sociales municipales en órganos intermedios participativos y en el territorio Gestionar los Servicios Sociales de Atención Primaria con organismos públicos autónomos. Los institutos son instrumentos adecuados de desconcentración de la gestión de las competencias de Servicios Sociales en el ámbito municipal, favorecen la transparencia y agilidad en la gestión económica, técnica y administrativa de los asuntos sociales y permiten incorporar mecanismos participativos. 6.1.6. Mejorar la representatividad y diversidad de actores participantes en los consejos e institutos Romper la dicotomía entre elevada representatividad social versus eficacia en la gestión de 154 Gobernanza de los Servicios Sociales municipales... los asuntos públicos. Para ello, se sugiere la articulación de mesas de trabajo o consejos sectoriales con competencias delegadas y que permitan la adopción de acuerdos en órganos intermedios representativos de cada colectivo o problemática social concreta, que represente al conjunto de entidades y organizaciones vinculadas con cada sector de población. Considerando el vertiginoso crecimiento de las asociaciones y otras entidades sociales, parece conveniente incluir en los estatutos o reglamentos la flexibilidad de incorporar a nuevos miembros sin necesidad de modificar éstos, simplemente mediante acuerdos al respecto. Establecer condiciones y garantías de representación de los miembros elegidos al efecto. Se trata que los representantes se comporten como tales: recogiendo previamente demandas y necesidades y trasladándolas adecuadamente, informando continuamente. Incorporar en los consejos e institutos la diversidad social, integrando con mayor intensidad a grupos sociales que en la actualidad se encuentran infra representados. 6.1.7. Generar estrategias relacionales de mayor impacto en las políticas sociales locales Clarificar objetivos y funciones de los consejos e institutos respecto de su incidencia directa en la política local, especificando los asuntos que ofrezcan un contenido real a estos mecanismos y, por tanto, motivar para participar. Se trata de innovar en el diseño e implementación de metodologías participativas que faciliten la participación real y evitar «caer» en la «inercia» de la no participación. Para ello se considera oportuno, compatibilizar mecanismos de participación asociativa con formas que promuevan la participación directa; vitalizar el capital social local, socializar y educar para la participación, impulsar y apoyar el asociacionismo y movimientos cooperativos, promover relaciones asociativas, generar redes sociales locales, apoyar acuerdos estratégicos entre organizaciones sociales y la Administración Local; coordinar centros y servicios locales, fomentar la co-gestión con las organizaciones vecinales y sociales de centros municipales; optimizar recursos, impulsar medidas de cooperación social entre organizaciones y la Administración Local, dotar con recursos municipales a los órganos de particiCuadernos de Trabajo Social Vol. 25-1 (2012) 143-158 Enrique Pastor Seller pación ciudadana existentes, generar espacios de encuentro entre administraciones, etc. 6.2. Aportaciones para profundizar en prácticas participativas orientadas a generar un dialogo significante en el ámbito local Las dimensiones a considerar (Pastor, 2010) para intensificar la participación ciudadana en los procesos, prácticas, actores y resultados pueden sintetizarse del siguiente modo: 6.2.1. Preparación adecuada Los contextos institucionales moldean significativamente los roles que adoptan los actores, los procesos y, por tanto, los resultados y la satisfacción de los actores. De ahí la necesidad de una coherencia en la organización municipal en escuchar y vincular los asuntos y acuerdos a la acción de gobierno. Por tanto, se precisa: estructura administrativa, desarrollo normativo, recursos de apoyo a organización y desarrollo y voluntad política. Una responsabilidad definida, un liderazgo sólido, que vincule la acción de gobierno local en su conjunto. 6.2.2. Planificación inclusiva y criterios de proceso Supone acordar unas «reglas de juego»: claridad de contexto, finalidad, propósitos y diseño en procesos y tareas. Deben cuidarse los ritmos, lugares de realización y criterios y selección de actores que han de participar. Los mecanismos de participación existentes en un gobierno deben estar conexionados entre sí, con el fin de dar una visión integradora de ciudadanía. El proceso participativo debe satisfacer lo emocional (que los participantes y la ciudadanía se sientan reconocidos, respetados y escuchados); el interés legítimo de los participantes que obtienen resultados y sentir, percibir y objetivar que el procedimiento es legítimo, justo, valioso. Estas cuestiones requieren evitar la improvisación, la fatiga del diálogo y los «simulacros». 6.2.3. Inclusión y diversidad de actores/territorios Supone la incorporación equitativa de actores, no sólo desde la representatividad estadística sino especialmente de la integración de todas las posiciones y voces. Ello requiere la igualdad en consideración y reconocimienCuadernos de Trabajo Social Vol. 25-1 (2012) 143-158 Gobernanza de los Servicios Sociales municipales... to en los debates desde una comunicación horizontal. Los criterios y procesos de selección de actores deben ser transparentes, evitando privilegiar a las «grandes corporaciones», ignorar a los opositores o bien privilegiar en exceso a los «expertos». La máxima inclusión y representatividad implica que la ciudadanía, en general, tenga la posibilidad de acceder e implicarse en las decisiones políticas y no solamente las personas y grupos cualificados. No resultan eficaces los mecanismos y procedimientos que excluyen de los procesos de toma de decisión a las personas o grupos por falta del «capital» (social, cultural, económico, tecnológico). 6.2.4. Equipo dinamizador y dinámica facilitadora Los facilitadores deben ser imparciales y cualificados, su tarea principal es cultivar una sensación compartida de apropiación, «empoderando» a la ciudadanía mediante un diálogo significante. Esto requiere: promover la indagación, la transparencia comunicativa y la autorreflexión. Ingredientes que potenciaran la comunicación y generarán un ambiente de confianza orientado a identificar coincidencias y diferencias argumentadas. En este sentido cabe incidir en que participar no es solo asistir a reuniones, sino apropiarse de los procesos de toma de decisiones mediante los mecanismos y medios diferentes de que disponemos, para escuchar y dar salida a la voz de los ciudadanos individualmente considerados, como grupos no organizados y como entidades sociales. 6.2.5. Dinámica motivadora Los mecanismos deben motivar y estimular para participar. Los participantes deben tener la sensación y certeza de que sus esfuerzos van en serio y que sus propuestas no se quedan en «papel mojado», sino que van a ser tomadas en consideración a la hora de tomar las respectivas decisiones. Las experiencias muestran el riesgo de utilizar los mecanismos para justificar decisiones adoptadas previamente, instrumentalizándose la opinión pública. 6.2.6. Colaboración y propósito compartido Supone trabajar en la planificación, recogida y presentación de información, etc., evitando promover opciones principales y secundarias. 155 Enrique Pastor Seller 6.2.7. Apertura y aprendizaje Ayudar a escucharse, explorar nuevas ideas sin restricciones, generar nuevas opciones; mediante un espacio de aprendizaje, distensión, autorreflexión. Para ello es importante cuidar ritmos, generar momentos de inflexión que permita a los participantes mostrarse vulnerables y hacerse vulnerables y permeables al resto de actores. 6.2.8. Funcionamiento deliberativo El proceso metodológico de las sesiones debe promover la deliberación. Los participantes no deben discutir sobre la base de la información que traen, sino que deben disponer de información extensa y diversa sobre los pros y contras, los diferentes intereses y opciones de la cuestión a tratar. Se trata de plantear todos los aspectos de un tema, hacerlo transparente, presentar los diferentes intereses, las soluciones alternativas, los costes y las repercusiones sobre la resolución de otros temas (ranking de prioridades), como sobre las personas afectadas. Es preciso transferir esas capacidades a la ciudadanía para que esta disponga del capital y poder para adoptar decisiones autónomas. Pero el input de información no se debe confundir con su utilización para conseguir el apoyo de la ciudadanía a una decisión adoptada por la Administración. 6.2.9. Transparencia y confianza La información debe ser accesible, multidireccional y policéntrica, y proceder de diferentes medios. Se trata de generar procesos claros y flexibles, tanto en la información como en la recogida de opciones, análisis de las mismas y generación de alternativas analizadas desde la viabilidad y la perspectiva de futuro. 6.2.10. Apoyo del bien general Los participantes deben apoyar el bien común, es decir la adopción de decisiones importantes para el futuro de la comunidad y no solamente de determinados grupos. Ello requiere de un diagnóstico comunitario basado en criterios de prioridades, adoptados de manera participativa. 6.2.11. Impacto y acción Hay que asegurarse de que cada esfuerzo participativo tiene potencial real, lo que implica 156 Gobernanza de los Servicios Sociales municipales... contemplar mecanismos de participación interconectados, adecuados, efectivos y capacitantes. Los participantes deben evidenciar que su participación es significativa, influye, impacta, con una sensación de apropiación real sobre procesos, tareas y decisiones. 6.2.12. Compromiso sostenidos y cultura participativa Los procesos participativos deben vincularse entre sí, con el fin de ir generando una cultura participativa en el contexto local que involucre todos los ámbitos de la vida cotidiana y a los agentes socializantes, en orden a generar una ciudad participativa. El análisis constata que los procesos participativos deben: — Ocuparse de la complejidad: social (perspectivas e intereses diversos), dinámica (causas y efectos sin tiempo y espacio) y generativa (futuro incierto o indeterminado). — Coordinar significados o entendimientos diversos: confianza y aceptación mutua, lo que implica transparencia de procesos y resultados. — Producir innovación: respuestas novedosas, diferentes y tangibles a la ciudadanía. — Posibilitar la deliberación: dialogar, sistematizar opiniones, encuadrar opciones alternativas y adoptar decisiones firmes acordadas. — Diálogo sostenido: producir procesos o resultados sostenibles a largo plazo. — Generar capacidades: empoderar, capacitar, crear grupos de trabajo, generar y apoyar lideres y su rotación, etc. En resumen, la transparencia e innovación real y efectiva de la participación ciudadana en el diseño, gestión y evaluación de las políticas de Servicios Sociales, se configura como elemento esencial para generar/reforzar/reconstruir la calidad democrática en el nivel territorial y organizacional más próximo. Su incorporación permitirá mejorar la eficacia y eficiencia de las políticas públicas, la prestación de los Servicios Sociales, adoptar decisiones significativas y vinculantes para los actores sociales y usuarios de centros/servicios, provocando una progresiva vitalización del capital social de los municipios y organizaciones sociales donde trabajamos desde las sinergias transaccionales. Cuadernos de Trabajo Social Vol. 25-1 (2012) 143-158 Enrique Pastor Seller Gobernanza de los Servicios Sociales municipales... 7. Referencias bibliográficas Alguacil, J. (2008). Espacio público y espacio político. La ciudad como el lugar para las estrategias de participación. Revista Académica de la Universidad Bolivariana, 20. Brugué, J., Font, J. y Gomà, R. (2003). Participación y democracia. Asociaciones y poder local. En M. J. Funes y A. Adell, Movimientos sociales: cambio social y participación. Madrid: UNED. Brugué, Q. y Vallés, J. M. (2005). Nuevos Ayuntamientos, Concejales Diferentes. Del gobierno de las Instituciones al Gobierno de las Redes.Madrid: FEMP. Casado, D. (coord.) (2010). Leyes de Servicios Sociales del Siglo XXI. Madrid: Cáritas-FOESSA. Colino, C. y Del Pino, E. (2008). Democracia participativa en el nivel local: debates y experiencias en Europa. Revista catalana de dret públic, 37, 247-283. FEMP (2002). Encuesta sobre la participación ciudadana en los Ayuntamientos. Madrid: FEMP. Font, J. (2001). Ciudadanos y decisiones políticas. Barcelona: Ariel. Font, J.; San Martin, J. y Schneider, C. (2006).Asociaciones y democracia: contribuciones y causalidades. En J. R. Montero, J. Font y M. Torcal (eds.), Ciudadanos, asociaciones y participación en España (pp. 241- 259). Madrid: CIS. Ganuza, E. y Francés, F. J. (2008). ¿A qué llamamos participar en democracia?: Diferencias y similitudes en las formas de participación. Revista Internacional de Sociología, LXVI(49), 89-113. Gutiérrez, M. (2005). La participación en los servicios públicos de bienestar. Un análisis del poder de los usuarios en el ámbito de la salud, la educación y los Servicios Sociales. Madrid: CES. Held, D. (2001). Modelos de democracia. Madrid: Alianza Editorial. 3ª ed. Herrera, M. R. (2010). Dramatización de la contienda política: acción colectiva y protesta en Argentina (1998-2005). (Tesis inédita). Universidad Pablo Olavide. Sevilla. INAP (2008). Evaluación de la participación pública en la elaboración de las políticas públicas. Madrid: Ministerio de Administraciones Públicas. Jorba, L. y Anduiza, E. (2009). Por qué y cómo evaluar la participación. En: M. Parés (coord.), Participación y calidad democrática. Evaluando las nuevas formas de democracia participativa (pp. 139-166). Barcelona: Ariel. Löffler, E. (diciembre, 2004). Experiencias internacionales de participación ciudadana en Europa. Trabajo presentado en I Congreso Internacional de Democracia Participativa. Madrid. Montero, J. R., Font, J. y Torcal, M. (ed.) (2006). Ciudadanos, asociaciones y participación en España. Madrid: CIS. Morales, L. (2005). ¿Existe una crisis participativa? La evolución de la participación política y el asociacionismo en España. Revista Española de Ciencia Política, 13, 51-87. Navarro, C., J.(2008). Participación Local. (Estudio n. 2.661). Madrid: CIS. Navarro, C. J., Cuesta, M., Font, J. (2009). ¿Municipios participativos? Participación política y ciudadanía en ciudades medias españolas. Madrid: CIS. Navarro, C. J. y Juarista, P., (2006). Funciones, actividades y facilitación pública de las asociaciones». En J. R. Montero, J. Font y M. Torcal (ed.), Ciudadanos, asociaciones y participación en España (pp. 223-240) Madrid: CIS. Pastor, E. (2009a). Participación ciudadana y gestión de políticas sociales municipales. Saarbrücken: Lambert Academic Publishin. Cuadernos de Trabajo Social Vol. 25-1 (2012) 143-158 157 Enrique Pastor Seller Gobernanza de los Servicios Sociales municipales... Pindado, F. (2008). La participación ciudadana en el ámbito local. Madrid: FEMP. Putnam, R. D. 2003). El declive del capital social. Un estudio internacional sobre las sociedades y el sentido comunitario. Barcelona: Galaxia Gutemberg-Circulo de Lectores. Rodríguez C., G. (junio, 2011). Políticas de atención a la dependencia en los regimenes de bienestar de la Unión Europea. Trabajo presentado en el Encuentro de Intervención y Políticas Sociales de Caja Madrid y X Jornadas de SIPOSO: Protección a la dependencia y leyes de Servicios Sociales del siglo XXI. Madrid. Rodríguez, G., Arriba, A., Marban, V. y Salido, O. (2005). Actores sociales y reformas del bienestar. Madrid: CSIC. Rodríguez Herrero, G. y Ajangiz, R. (2007). Descentralización municipal y participación ciudadana: evaluación de una experiencia consolidada. Revista Española de Ciencia Política, 17, 181-197. Roque, V. y Zimermann, V.J. (2009). Descentralização na gestão pública e estruturas subnacionais de gestão do desenvolvimento: o papel dos consorcios intermunicipais. Revista Brasileira de Gestão e Desenvolvimento Regional 5, (3), 3-28. Ruiz, J.I. (dir). (2006). El sector no lucrativo en España. Una visión reciente. Bilbao: Fundación BBVA. Subirats, J. (2007). Los Servicios Sociales de Atención Primaria ante el cambio social. Madrid: Ministerio de Trabajo y Asuntos Sociales. Tarrow, S. (1997). El Poder en Movimiento: los nuevos movimientos sociales, la acción colectiva y la política. Madrid: Alianza Universidad. Tilly, C. (2008). Contentious Performances. Cambridge: Cambridge University Press. Tilly, Ch. y Tarrow, S. (2006). Contentious Politics. Boulder (Co): Paradigm Publishers. Vilá, T. (junio, 2011). Las leyes de Servicios Sociales del siglo XXI. Análisis jurídico-administrativo. Trabajo presentado en el Encuentro de Intervención y Políticas Sociales de Caja Madrid y X Jornadas de SIPOSO: Protección a la dependencia y leyes de Servicios Sociales del siglo XXI. Madrid. Warren, M. E., (2001). Democracy and association. Princeton: Princeton University Presss. Zurbriggen, C. (2011). Gobernanza: una mirada desde América Latina. Revista Perfiles Latinoamericanos, 39, 63-79. 158 Cuadernos de Trabajo Social Vol. 25-1 (2012) 143-158 

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1211 Sensors and Materials, Vol. 35, No. 4 (2023) 1211–1220 MYU Tokyo S & M 3240 Development of Earthquake Detection and Warning System Based on Sensors Fu-Hsien Chen, Horng-Lin Shieh,* and Jih-Fu Tu Lunghwa University of Science and Technology, No. 300, Sec. 1, Wanshou Rd., Guishan District, Taoyuan City 333326, Taiwan (Received September 12, 2022; accepted March 14, 2023) Keywords: earthquake, embedded system, sensors, ADXL345, air quality Earthquakes often cause severe disasters. Taiwan is located in an earthquake zone, where earthquakes occur frequently. For example, the Meinong earthquake in Kaohsiung in 2016 and the Hualien earthquake in 2018 caused the collapse of buildings in Tainan and Hualien, resulting in a total of 232 deaths and more than 100 injuries. Due to the strong vibration, earthquakes are often accompanied by fire and the leakage of toxic gases, causing loss of life and property. In this study, we integrate Arduino, sensors, and transmission technology to design an earthquake detection and warning system. When an earthquake is detected, the system immediately notifies everyone to evacuate. When the harmful gas concentration exceeds a critical value, the warning light is switched on, and the exhaust fan is turned on to extract the indoor toxic gas. When the flame sensor detects a flame, the system activates an alarm to warn people to escape quickly. In the designed system, capacitive three-axis accelerators are used as vibration measurement sensors to issue warnings in the early stage of an earthquake. In addition, the system includes an IR flame sensor to detect fires and an MQ series air quality sensor to detect harmful gases. The newly designed sensing system not only has the advantage of notifying people immediately during an earthquake but is also cheap and easy to use. 1. Introduction Taiwan experiences frequent seismic activity due to the ongoing subduction of the Philippine and Eurasian plates.(1) Old buildings are often cracked and tilted due to earthquakes. As shown in Fig. 1(a),(2) the 2016 Meinong earthquake in Kaohsiung caused a building to collapse in Yongkang District, Tainan City, killing 115 people. Figure 1(b) shows a building that collapsed during the Hualien earthquake in 2018.(3) This earthquake was the most severe in Taiwan since the Kaohsiung Meinong earthquake in 2016. The quake caused four buildings to collapse and 17 people to die. Earthquakes are often accompanied by fires and toxic gas leakage. Therefore, their detection is also necessary when an earthquake occurs. In this study, we integrate embedded systems, wireless transmission technology, network access, and sensors to design an earthquake-sensing system. When an earthquake is detected, *Corresponding author: e-mail: shieh@gm.lhu.edu.tw https://doi.org/10.18494/SAM4116 ISSN 0914-4935 © MYU K.K. https://myukk.org/ 1212 Sensors and Materials, Vol. 35, No. 4 (2023) (a) (b) Fig. 1. (Color online) (a) Building collapse caused by the Kaohsiung Meinong earthquake. (b) Building collapse caused by the Hualien earthquake in Taiwan. the system warns users with a buzzer and notifies people immediately of a tilted building, harmful gas leakage, or a fire. The designed system includes Wi-Fi wireless transmission combined with a three-axis accelerator, an MQ series sensor, and a flame sensor to detect earthquakes, harmful gases, and fires, respectively. The system can not only warn users to escape early but also detect possible threats to life when earthquakes occur. 2. Literature Review Hsu(4) proposed a wireless earthquake early warning and detection system that used a threeaxis accelerometer to detect earthquakes. In the system, temperature-sensitive changes were avoided by using capacitors with direct current (DC) to reduce noise. The sensing signal of the three-axis accelerometer was transmitted through the serial peripheral interface (SPI). It then sent the signal to the server using a wireless network, and a data analysis method was used to notify the user of the risk. The advantage of the system is the use of DC capacitors to reduce the noise density of the three-axis accelerometer. However, it only detects earthquakes and does not warn users of building tilt, harmful gases, and fires caused by earthquakes. Hsu and Sheu(5) proposed an ultralow-complexity algorithm for an earthquake early warning system. The algorithm used the moving average method to eliminate short-term noise and collected the turning points where the earthquake starts to be detected as important feature points for earthquake identification. Then, the quadratic differentiation method was used to differentiate the turning points and calculate the earthquake’s magnitude. The primary purpose of their study was to quickly and accurately calculate the magnitude of earthquakes. Sensors and Materials, Vol. 35, No. 4 (2023) 1213 Yoon et al.(6) developed a fingerprint and similarity thresholding algorithm (FAST) to analyze seismic waves. FAST built a compressed fingerprint waveform based on the main identified features, stored similar fingerprints in a database to enable a quick search, and monitored a series of earthquake detections. Jayron et al.(7) developed an earthquake detector unit system using Arduino Mega and an ADXL335 accelerometer. In this system, when the Arduino Mega earthquake detector detected ground motion of a specified intensity level, the system triggered an alarm that made a sound. A solar panel system was integrated to power the designed system. Such a solar-power-operated earthquake detector with an automatic alarm system helped raise awareness when earthquakes occurred, reducing physical harm to humans and accidents. In the system, an LCD was used to display messages on the current status and readings of the ADXL335 accelerometer. Although its solar power supply reduced its environmental impact, it could not detect the harmful gases produced when an earthquake occurs. Grover and Sharma(8) defined information about an earthquake and designed a system to predict earthquakes. Their paper briefly introduced various types of seismic waves with their respective frequencies. Their developed system integrated an Arduino Uno microcontroller (ATMega 328) and an accelerometer (ADLX335) used to measure seismic data in earthquakeprone areas, which were connected to a data center by a wireless network. The ADLX335 is a small, thin, low-power, complete three-axis accelerometer with signal-conditioned voltage outputs(9) and can measure static and dynamic accelerations in the X, Y, and Z directions. The microcontroller converted this data into a digital form, then sent it wirelessly to the data center. If there is no earthquake, the system is in a measurement loop. If the system predicts an earthquake, the system produces a warning sound to users. Faiazuddin et al.(10) combined the IoT, a Raspberry Pi 4 device, Arduino Uno, sensors, and ThingSpeak cloud technology to build an indoor air quality monitoring system. The system mainly detects indoor CO2, CO, NO2, H2, NH3, and other gases. The relevant measurement data is sent to the cloud for MySQL storage and analyzed in real-time with ThingSpeak. The monitoring system builds a module for calculating the air quality index (AQI), which collects information from various sensor network nodes to increase the number of network nodes deployed and more accurately measure the air quality. Liu et al.(11) implemented a low-cost and practical mobile indoor air quality monitoring system using Arduino and sensors. They used tracking and obstacle avoidance sensors to achieve autonomous movement and gas sensors for indoor air quality monitoring. The system collected data for CO, PM2.5, temperature, and humidity with the framework and professional measurement instruments. The experimental results showed that the errors for the CO and PM2.5 concentrations, temperature, and humidity were only 0.086, 0.81, 0.15, and 1.2%, respectively. It was found that the accuracy with Arduino and the sensors was similar to that of professional instruments. Schieweck et al.(12) integrated the concept of smart homes with the efficient use of energy and ventilation technology in low-energy buildings. They analyzed in detail the impact of environmental parameters measured in Germany and Sweden on indoor air quality, personal thermal comfort, and occupancy behavior in smart homes. They also introduced sensor 1214 Sensors and Materials, Vol. 35, No. 4 (2023) technology to assess the level of pollution. As different types of sensing technologies are suitable for different situations, they evaluated available sensor technologies, discussing the application of sensors and their limits and possibilities for monitoring the concentration of particles and gaseous pollutants indoors. 3. System Architecture Figure 2 shows the architecture of the proposed system. The server, which is the data center, is used to set up the database system required for the design and store sensor data and serves as a monitoring interface to display data and respond to queries. The embedded system uses Arduino as its core. The sensing modules in the system are seismic and building tilt sensor modules, which use an ADXL345 three-axis accelerometer. Smoke, gases, and air quality are respectively sensed by MQ-2, MQ-5, and MQ-135 sensors, and a flame is detected by an IR flame sensor. 3.1 User interface The user interface includes functions such as user login, wireless communication operation, database query, data statistics, and a hazard alarm, as follows: A. User login: Responsible for creating user accounts, entering user passwords, and verifying database access rights. B. Wireless communication operation: Responsible for establishing a wireless connection between the server and the Wi-Fi router, including the IP address and port setting. C. Database query: Various data queries, including air quality, building inclination, and flame information. D. Data statistics: The data for a week, a month, or the whole year is presented in a graphical interface to facilitate inquiries from users about environmental changes. E. Hazard alarm: When an earthquake, gas leakage, or fire occurs, or the building is tilted beyond a certain angle, the system issues a warning sound to alert users of the problem. Fig. 2. (Color online) Architecture of proposed system. Sensors and Materials, Vol. 35, No. 4 (2023) 3.2 1215 ADXL345 three-axis accelerometer The accelerometer calculates the acceleration in three dimensions.(13) The proposed system uses an ADXL345 three-axis accelerometer for earthquake sensing and building tilt detection, which is a thin, short gravity sensor with low power consumption. It consists of fixed and moving plates that deflect in any direction subject to acceleration in the x, y, and z directions.(14) Its digital output data is in the format of a 16-bit binary code, which is transmitted with an SPI or I2C digital serial transfer. It is ideal for static acceleration or center-of-gravity tilt sensing applications, and its high resolution (4 mg/LSB) enables the measurement of changes in the tilt of less than 1.0°. Figure 3 shows the diagram of the tilt angles for ADXL345. The following angles are calculated as(13) α1 = tan −1   Ax  ,  Ay2 + Az2     Ay (1)  ,  A2 + A2  x z   β1 = tan −1   Az γ 1 = tan  2  Ax + Ay2  −1 (2)  ,   (3) where Ax, Ay, and Az represent the acceleration values in the respective directions of the coordinate axes, while α1, β1, and γ1 represent the angles between each acceleration vector and the horizontal line. Z Y ADXL345 X γ1 α1 Fig. 3. β1 (Color online) Tilt angle diagram for ADXL345. Horizontal line 1216 3.3 Sensors and Materials, Vol. 35, No. 4 (2023) IR flame module The IR flame sensing module uses IR rays that are sensitive to flames and IR receiver tubes to detect the presence of fire, and then converts the brightness of the flames into a voltage signal output. The critical characteristics of the flame model are as follows. A. The module detects light sources whose flame wavelength ranges from 760 to 1100 nm.(15) The larger the flame, the longer the test distance. B. The detection angle is about 60° and is not sensitive to flame light. C. The sensitivity is adjusted with an adjustable precision potentiometer. D. The working voltage is 3.3–5 V. E. The voltage increases with the distance from the flame source. The output voltage in the case of no fire source is 4.8 V. When a fire occurs, the voltage value read by Arduino is lower than 4.0 V, and the system reports a suspected fire. The statistics of the relationship between distance from fire source and output voltage are presented in Table 1. 3.4 Air quality sensor module In this study, the air quality is monitored using MQ series sensors. These sensors detect six different gases, namely, methane, propane, butane, smoke, carbon monoxide, and ammonia. They have a fine porous stainless steel mesh for rapid heat transfer to prevent a gas explosion, and they use tin dioxide (SnO2) as a gas-sensing material as it has low conductivity in clean air. When the concentration of harmful gases in the air increases, the conductivity of the sensor increases, and then a simple ADC conversion circuit converts the change in conductivity into an output signal corresponding to the gas concentration. MQ sensors are classified as follows. MQ-2: Smoke gas sensor. MQ-3: Alcohol sensor. MQ-4: Methane gas sensor. MQ-5: Liquefied gas/methane sensor. MQ-6: Propane/liquefied gas sensor. MQ-7: Carbon monoxide/combustible gas sensor. MQ-8: Hydrogen gas sensor. Table 1 Relationship between distance from fire source and output voltage. Distance (mm) Output (V) Distance (mm) 10 0.06 20 40 0.09 60 80 0.11 100 200 0.18 300 400 0.22 500 600 0.30 700 800 0.43 900 1000 0.70 2000 8000 3.52 — Output (V) 0.07 0.1 0.13 0.19 0.25 0.32 0.50 1.44 — Sensors and Materials, Vol. 35, No. 4 (2023) 1217 MQ-9: Carbon monoxide/methane/liquefied gas sensor. MQ-135: Air pollution/harmful gas sensor. Because of the impact of air quality at home, we use four types of sensors: MQ-2, MQ-5, MQ-7, and MQ-135. 4. Experimental Results Figure 4(a) shows the front view of the house model built for this study. Sensors are installed in the upper left corner of the house. The Arduino and three-axis accelerometer are located at the bottom of the model, and the wireless Wi-Fi router is placed behind the model house as shown in Fig. 4(b). Figure 4(c) shows the user interface, where “IP” displays the IP address of the house model and “3-axis accelerometer” displays the angles α1, β1, and γ1 measured by the three-axis accelerometer. The menus “Smoke”, “Air quality”, “Gas”, and “Flame” display the indoor smoke quantity, air quality, gas concentration, and fire monitoring value, respectively. The graphs on the right of each monitoring item display the monitoring status, where the blue line indicates the monitoring value and the red line indicates the critical value. When the system detects a monitoring value exceeding the threshold, it generates a warning sound and displays the sign “Warning message”. The “Start” button in Fig. 4(c) is used to start the system, the “End” button is used to stop the system, and the “View” button is used to view the log of abnormal events. Figure 5(a) shows how the up, down, left, and right displacements in an earthquake were simulated. Figure 5(b) shows the values measured by the three-axis accelerometer. The values of 0.11, −0.29, and 0.79 indicate an earthquake, and the system sounds an alarm and displays a warning message. To simulate gas leakage, we use a gas lighter to generate gas at a distance of about 5 cm from the sensor as shown in Fig. 6(a). As shown by the blue line in Fig. 6(b), when the system detects gas with a concentration exceeding a threshold value, a warning message is displayed to inform the user of a gas leakage. Figure 7(a) shows the fire used for testing. We use a gas lighter to generate a flame about 5 cm from the sensor. Figure 7(b) shows that when a fire is generated, the sensor immediately detects it and displays a fire alarm message. It also generates a warning sound. In the figure, the blue line shows the value for the flame and the red line shows the threshold value. (a) Fig. 4. (b) (c) (Color online) (a) House model built for this study. (b) Wi-Fi router. (c) Human–machine interface. 1218 Sensors and Materials, Vol. 35, No. 4 (2023) (a) (b) Fig. 5. (Color online) (a) Simulation of an earthquake. (b) Warning message generated when the sensor detects an earthquake. (a) (b) Fig. 6. (Color online) (a) Gas-sensing experiment. (b) Warning message generated by the system when the sensor detects gas. (a) (b) Fig. 7. (Color online) (a) Fire experiment. (b) Warning message generated when the sensor detects a fire. A warning sound is also generated. Next, we generate smoke by burning tissue paper as shown in Fig. 8(a). When smoke is generated indoors, the smoke detection line in blue exceeds the red line indicating the threshold value, and the system generates a warning sound and message. When there is an earthquake, gas, or fire in the room, the system records the event along with the time of occurrence in the back-end server system through the wireless network. As shown in Fig. 9, when the user presses “View”, the system displays all abnormal events, which can be queried. Sensors and Materials, Vol. 35, No. 4 (2023) (a) Fig. 8. 1219 (b) (Color online) (a) Smoke experiment. (b) Warning message generated for smoke. Fig. 9. (Color online) Query of abnormal events in the database. Table 2 Comparison of system in this study with other systems. Jayron et al.(7) Grover and Sharma(8) Earthquake detection √ √ Warning sound √ √ Harmful gas detection × × Flame detection × × Data center × √ Proposed method √ √ √ √ √ Table 2 shows a comparison of the system designed in this study with other systems. A large earthquake causing damage to buildings is often accompanied by fires and toxic gas leakage. Therefore, it is necessary to detect earthquakes, fire, and toxic gas concentrations at the same time. Our newly designed system detects all three, whereas the systems designed by Jayron et al.(7) and Grover and Sharma(8) detect earthquakes but not fire and toxic gases. To detect possible fires and abnormal toxic gas concentrations when an earthquake occurs, our system stores the data in a cloud database through wireless transmission for subsequent inquiries. The system designed by Grover and Sharma included a data center to store seismic data but lacked information on fire and toxic gases. The system developed by Jayron et al. did not include a data center. Therefore, our system has greater functionality than the other systems. 1220 5. Sensors and Materials, Vol. 35, No. 4 (2023) Conclusions When an earthquake occurs, the time available to find a sheltered space is only a few to a dozen seconds. Therefore, in the early stages of an earthquake, it is important to provide early warnings to people to seek shelter as soon as possible. To warn people of an earthquake, harmful gas, or fire so that they can evacuate effectively, we established an earthquake disaster prevention system combining Arduino, sensors, and 4G transmission technology. The results of testing the system showed that it functions appropriately. In the future, the system will require modularization and packaging so that it can be applied as a disaster prevention system in houses, businesses, factories, public offices, and schools. The designed system provides the basis for a large-scale application to warn people of earthquakes and accompanying fires and gas leakage. Acknowledgments This research was funded by the National Science and Technology Council, Taiwan (grant number MOST 111-2637-E-262-001) and the MOE Teaching Practice Research Program (grant number PSK1110219). References 1 Y. M. Wu and H. Mittal: Sensors 21 (2021) 1. https://www.mdpi.com/1424-8220/21/22/7649 2 S. Mori, C. W Lu, and M. H. 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The International Journal of Life Cycle Assessment (2023) 28:554–565 https://doi.org/10.1007/s11367-023-02154-0 LCA OF WASTE MANAGEMENT SYSTEMS Use of life cycle assessment to evaluate circular economy business models in the case of Li‑ion battery remanufacturing Benedikte Wrålsen1 · Reyn O’Born2 Received: 11 October 2022 / Accepted: 1 March 2023 / Published online: 15 March 2023 © The Author(s) 2023 Abstract Purpose The purpose of this study is to advance and illustrate how life cycle assessment (LCA) can assess circular economy business models for lithium-ion batteries to verify potential environmental benefits compared to linear business models. Scenarios for battery repurpose are assessed to support future decision-makers regarding the choice of new versus second life batteries for stationary energy storage. A procedure to determine the substitution coefficient for repurpose and reuse of batteries is proposed. Methods Two different circular economy business models are assessed by applying primary data from two Norwegian companies for the development of a new life cycle inventory. With this new data, the authors compare second life battery (from first life in electric vehicle) scenarios and avoided production potential by performing a complete consequential LCA. Building on earlier work, a procedure to identify the substitution coefficient (i.e., potential for avoided production) for battery life cycle assessments is proposed. Interviews during factory visits were performed to identify a technical and a market factor affecting the substitution coefficient. Results and discussion This study illustrates how life cycle assessment methodology can detect and thus enhance the potential environmental benefits and trade-offs of circular economy business models. Results show that the CBMs which use second life batteries correspond to 16% (for global warming potential) of manufacturing a new battery. This means that a second life battery must avoid > 16% production of a new battery to become the preferred alternative. Hence, circular economy business models with second life batteries can generate net environmental benefits while the remaining battery capacity and market price are identified factors that can alter the potential environmental benefits. The findings suggest that assumptions concerning the avoided production emissions are crucial for understanding the overall impacts of battery value chains. Conclusions Circular economy business models which enable second life batteries show lower environmental impacts compared to a new battery when it can partly avoid production of a new battery. Based on the identified technical and market factor affecting this potential, a key message to industry and other organizations is that second life batteries should be chosen over new batteries. This depends on the remaining capacity being satisfactory for the new application, and the investment is not performed because of a low price compared to a new battery. Consequential LCA practitioners adopting a market approach while evaluating battery reuse and repurpose should model and account for the avoided production potential. Keywords Circular economy · Life cycle assessment · Batteries · Substitution · Circular economy business models · Remanufacture Communicated by Xin Sun. * Benedikte Wrålsen benedikte.wralsen@uia.no Reyn O’Born reyn.oborn@uia.no 1 School of Business and Law, University of Agder, Grimstad, Norway 2 Faculty of Engineering and Science, University of Agder, Grimstad, Norway 13 Vol:.(1234567890) 1 Introduction Demand for stationary energy storage such as high-capacity batteries to support grids and store renewable energies is increasing (IEA 2020). Simultaneously, the electric vehicle (EV) market, powered by Li (lithium)-ion batteries (LIBs) is growing continuously (IEA 2021). This development in LIB demand increases the consumption of metals and other valuable materials used in battery production (BloombergNEF The International Journal of Life Cycle Assessment (2023) 28:554–565 2022) while used LIBs are increasingly seen as a future waste problem (Pehlken et al. 2017). These trends require intelligent management of used batteries and battery materials to ensure that EVs (and their LIBs) are sustainable in a life cycle perspective. Circular economy business models (CBMs) can contribute to tackle the upcoming wave of used batteries (Jiao and Evans 2016; Olsson et al. 2018). A CBM is a plan for how a business can operate profitably while ensuring decreased environmental impacts through closed-loop supply chains and reduced resource consumption (e.g., sharing models, energy efficiency, and applying recyclable materials in primary production) (Bocken et al. 2019; Geissdoerfer et al. 2020; Lüdeke-Freund et al. 2019). CBMs can be used to implement sustainable strategies to manage a product after its first life, such as repurposing in a second life application prior to recycling of the materials. However, current research questions the real advantages of such circular economy (CE) efforts and recognize trade-offs where further assessment is needed to understand how these CBMs affect the life cycle environmental impacts of product systems (Manninen et al. 2018; Rigamonti et al. 2017; Saidani et al. 2019; Zink and Geyer 2017). Life cycle assessment (LCA) is a useful methodology to assess CE efforts, where consequential LCA methods can be used to determine if CBMs should be implemented (Haupt and Hellweg 2019; Ncube et al. 2022; Peña et al. 2021; Rigamonti and Mancini 2021; Stucki et al. 2021; van Loon et al. 2021). Consequential LCA aims to detect environmental consequences of future decisions (Frischknecht et al. 2017; Schulz-Mönninghoff et al. 2021), such as a choice between new business models (Løkke et al. 2020). LCA can thus be a valuable tool to support sustainable, circular business opportunities (Murakami et al. 2019). Earlier LCA studies (Ahmadi et al. 2017, 2014; Bobba et al. 2018; Commission et al. 2018; Cusenza et al. 2019; Ioakimidis et al. 2019; Kamath et al. 2020a, b; Philippot et al. 2022; Richa et al. 2017; Wang et al. 2022; Wilson et al. 2021; Xiong et al. 2020) found that second life batteries have lower environmental impacts compared to new batteries. However, these studies did not include primary, new inventory data for the remanufacturing processes required to form a second life battery from an EV battery pack to a larger battery for a stationary energy storage applications as most LCA studies of LIBs rely on secondary data (Aichberger and Jungmeier 2020). Schulz-Mönninghoff and colleagues (2021) assessed repurposing of a battery pack, however, only included testing, calibration, software updates, and shipment to assembly site for the reassembly life stage in Germany. The study included new inventory data for the installation, however, not from battery module level (i.e., not dismantling the battery pack and reassembly the module parts for a second life battery pack). Current research on LCA of second 555 life batteries does not currently have focus on the disassembly and remanufacturing processes. In consequential LCA (cLCA), determining a precise substitution coefficient (i.e., the amount of avoided production due to use of a second life product or material) is crucial for life cycle impact assessment results (Chalmers et al. 2015). Despite this, several consequential LCA studies on waste management assume one hundred percent avoided production (causing negative emissions) for second life products and recycled materials, which is seldom the case (Heijungs and Guinée 2007; Rigamonti et al. 2020; Zink et al. 2016; Zink and Geyer 2017). There is an ongoing discussion on how to determine the substitution coefficient in LCA studies (Vadenbo et al. 2017). Rigamonti and colleagues (2020) suggest a guideline to develop the technical substitution coefficient, representing degree of technical replacement potential. The researchers encourage LCA practitioners to develop coefficients for other secondary materials or products to advance and harmonize their work. Market-related factors also affect the substitution coefficient, such as price mechanisms (Zink et al. 2016). Increased emphasis is needed on considering market characteristics such as substitution, rebound, and price effects in cLCA (Yang and Heijungs 2018). The authors are not familiar with other LCA studies that propose a technique to combine a technical and market factor to identify the substitution coefficient for batteries. This study assesses the consequential environmental impacts between two different circular economy business model alternatives for second life LIBs based on two companies in Norway. These two CBMs utilizing used EV batteries for energy storage are compared to the existing linear business model using new LIBs. The first CBM enables repurposing of used EV battery packs for a second life in stationary energy storage systems to obtain increased selfsufficiency (i.e., reduced grid dependence by local renewable energy generation). The second CBM enables repurposing by dismantling the pack into modules and thereafter reassembly of the modules to a second life battery pack. The origin of the batteries and the second life application for both CBMs are identical but differ in how these used batteries are dismantled and reassembled. A new life cycle inventory is introduced for the two CBM cases including the remanufacturing and installation processes required. Applying consequential LCA, this research investigates real environmental effects of CBMs where batteries are repurposed in a stationary energy system as identified in earlier work (Wrålsen et al. 2021). As part of the complete assessment, a procedure for practitioners to identify the substitution coefficient in battery reuse and repurpose cases is proposed, implementing both a market and a technical factor. The methodological advancements are illustrated within the two cases described in Sect. 3. 13 556 2 Li‑ion battery repurpose Previous research shows that remanufacturing and repurposing is identified as the CBM with the highest potential for LIBs (Wrålsen et al. 2021). Battery repurpose is when a used battery is applied in a different application than it was originally designed and manufactured for, for example, if a battery has a first life in an EV and a second life in a stationary energy storage system. LIBs have high energy density compared to other batteries and are recognized as interesting for repurpose when there is remaining capacity left after first life (Melin et al. 2021; Neubauer et al. 2015; Wind et al. 2021). If there is remaining capacity left depends on the case-specific use and application. Thus, the lifetime of second life batteries will vary (Wrålsen and Faessler 2022). A study testing the cycle lifetime of a used EV battery with unknown user history found that the battery could be charged and discharged 2033 cycles (Braco et al. 2020). In cases where repurposing is considered, the used batteries can be tested through characterization to assess remaining capacity and suitability for repurpose (Harper et al. 2019). Current CBMs which enable a second life remanufacture EV batteries at different levels, primarily (1) remanufacturing of the complete battery pack or (2) remanufacturing by first dismantling the pack to several modules and then reassembling a new pack based on these modules. This is possible as an EV battery pack consists of several connected battery modules. These modules consist of several battery cells. Since the commercialization of LIBs in 1991, researchers have worked to increase the energy density of the battery cells by testing different materials and compositions (Zhao et al. 2021). Several LIB chemistries are now in use in EVs. The longest (real) driving range in a commercialized EV is currently (in 2023) almost 700 km, according to EVDB (EV database 2023). Remanufacturing enabling repurpose will extend the battery lifetime before the materials are recycled at the final end-of-life. Recycling technologies for LIBs are underdeveloped and repurpose will enable more time for LIB recycling technologies to improve (Kotak et al. 2021). These technologies must be improved as battery packs consist of several valuable materials, for example, aluminum, steel, copper, nickel, cobalt, and lithium. The two latter are the most critical in terms of reserves and supply risks (to some extent also nickel) (Xu et al. 2020). High-nickel batteries such as the lithium nickel manganese cobalt oxide (NMC)811 chemistry is a growing trend, reducing market share of lower nickel content chemistries such as NMC 111. Chemistries without cobalt is also a growing trend (Wind et al. 2021). Recognized battery repurpose applications can be grouped into (1) in-front-of-the-meter applications (i.e., the power passes through the meter before reaching the end-user), (2) 13 The International Journal of Life Cycle Assessment (2023) 28:554–565 behind-the-meter (i.e., the power can be used on-site), and (3) off-grid (i.e., battery systems not connected to the electricity grid). A second life battery can for example be used for energy arbitrage where the battery stores electrical energy when the electricity from the grid is cheap to purchase and uses this when the electricity price is high. This is an example of a repurpose application in-front-of-the-meter. The used battery with remaining capacity can alternatively be used for peak shaving to reduce the demand peaks which often cost more. This is an example of a repurpose application behind-themeter (Faessler 2021). A disadvantage of second life batteries, and a potential challenge with repurpose practice, is that the batteries are designed for their first use application (e.g., an EV) and are therefore not technically optimal for the second life application (Rallo et al. 2020; Reinhardt et al. 2019). 3 Case studies The life cycle inventory list in this study is based on two existing projects from two Norwegian companies working with different circular economy business models. Both projects apply used EV batteries for stationary energy storage systems for storing solar energy to increase self-sufficiency of electricity and decrease grid dependence. 3.1 Circular economy business model 1: Eco Stor Eco Stor AS was established in 2018 in Norway to commercialize stationary energy storage solutions based on second life batteries from electric vehicles (Eco Stor AS 2022). The company offers solutions for applications such as solar energy storage (increased self-consumption), peak shaving, grid infrastructure support, and demand side grid trading. Their subsidiary company in Germany uses new batteries in large-scale systems for grid support applications (linear business model), and in Norway, second life battery packs from electric vehicles are used (circular economy business model). The used battery packs are sent from central Europe to Eco Stor in Norway by their vehicle manufacture business partners. This car manufacturer characterizes and sorts the used batteries to ensure only quality battery packs with sufficient remaining battery capacity are repurposed. Repurposing used battery packs from EVs can be challenging due to the lack of data sharing from the battery management system (BMS). The BMS programming code and historic consumer data is currently protected by the owner to hinder hacking and to secure business value. This hinders third-party firms like Eco Stor from having a history of battery cycling and battery state of health, which is critical to understanding how the used batteries can be repurposed (Faessler 2021). Therefore, cooperation with an electric vehicle manufacturer and The International Journal of Life Cycle Assessment (2023) 28:554–565 BMS owner is key to success for this CBM. The life cycle inventory list built for CBM 1 (repurposing battery packs) in this study reflects the required resources to build generic racks with battery packs applied for stationary energy storage projects. For this case, the energy storage system consisted of a 280 kWh battery system. 3.2 Circular economy business model 2: Batteriretur Batteriretur is a Norwegian company approved to collect all types of used batteries in Norway, including high-capacity batteries such as lithium-ion batteries from EVs (Batteriretur 2022). The company is owned by several vehicle manufacturers and is responsible to treat the used batteries sustainably according to the European Union’s Battery Directive from 2006 (European Union 2006). This directive states that the actors distributing the battery on the market are responsible for providing a collection (take-back) system and to recycle it to the full extent possible. The end-of-life collection, discharge of pack, characterization, dismantling of pack, and further distribution are Batteriretur’s responsibility, and they do this on behalf of the battery market distributors. There are two options for the final distribution stage, as the batteries can either be sold to second life distributors or battery recyclers. Currently, most used batteries are sent for treatment and recycling in Europe. To analyze potential economic and environmental gains, Batteriretur developed a pilot project using second life battery modules (the main pack components) from EVs to store solar energy at their plant to reduce their dependency on the grid. The inventory built for CBM 2 (battery modules) in this study reflects the required resources for this pilot project. The case energy storage consisted of a 500 kWh battery system. 557 This study uses a consequential modeling approach that aims to achieve relevant information about environmental consequences to support prospective decisions and considers the consequences of decisions within the market (Ekvall and Weidema 2004; Yang and Heijungs 2018). The decision considered in this study is choosing a traditional, linear business model with a new LIB for stationary energy storage versus second life batteries based on two different CBMs. To account for the potential avoided production, substitution is included in the modelling. This method is preferred by the ISO 14044:2006 (ISO 2006) standard for LCA, where “negative” impacts from avoided production can be included (ISO 2006). CBM 1, CBM 2, and the new battery have the same function: 1 kWh of battery capacity ready for use in a stationary energy storage system application. It is crucial that these product systems are comparable to examine effects of substitution (Weidema 2000). Figure 1 shows the required processes for the two CBMs. Recycling of the materials is outside the scope of this study. The complete, unmodified battery pack is used in CBM 1, and thus, dismantling, characterization, sorting, and reassembly are not part of the scope for CBM 1. Dismantling and reassembly are not necessary because the complete battery pack from the EV is used in the second life battery pack. Characterization and sorting, checking the state-of-health of the battery packs, and sorting them are done before the battery packs are received by the case company. CBM 2 requires characterization and sorting which are assessed within the scope. As this CBM builds second life battery packs based on battery modules from EVs, the dismantling of the battery packs and reassembly of the modules are required processes. The manufacturing of the machinery and tools at the remanufacturing plant are not included within the scope of either CBMs. 4 Material and methods 4.2 Life cycle inventory data 4.1 Goal and scope The inventory data for each CBM was collected from the two case companies. Both companies were visited following a digital interview. The company visits and the digital interviews followed the same semi-structured interview guide to secure the most crucial information for the inventory while being open to additional relevant information. Before and after the interview, the authors had e-mail correspondences with the case companies discussing relevant processes and developed a basic data collection procedure for collecting the inventory data at each company. Background data is from the consequential Ecoinvent database v.3.8 (Wernet et al. 2016) where a global approach on material production was adapted, except for production of new LIBs, which was assumed to come from China. The major upstream supply for new batteries comes from China; although Europe and the USA are expected to increase their market share (IEA The goal of this LCA study is to assess the environmental consequences of two CBMs which utilize used EV battery packs (CBM 1) and modules (CBM 2) for battery energy storage systems. A secondary goal of this study is to compare these two CBMs with a new NMC 811 battery pack used for the same purpose. The two CBMs are assessed as part of three different scenarios where avoided production (potential for a second life battery to replace new production) is included. The scope includes the resources required to remanufacture a used EV LIB for a new life in a stationary energy storage system and installation in this new application. The functional unit of this study is 1 kWh capacity (second life) NMC 811 battery pack. The inventory is modelled in mass, where 1 kWh capacity NMC 811 battery pack weights 6.7 kg (Crenna et al. 2021). 13 558 The International Journal of Life Cycle Assessment (2023) 28:554–565 X kWh capacity new battery Characterization and sorting Install packs in rack CBM 1 Install racks in application 1 kWh capacity spent battery End-of-first-life battery pack CBM 2 Dismantling, characterization, sorting, and reassembly CBM 3 Install packs in rack Install racks in application Recycling of materials End-of-second-life battery pack Fig. 1  Bold border and gray area represent system boundaries: the scope includes circular economy business model (CBM) 1 and 2 for lithiumion batteries (LIBs). Stippled lines represent substitution potential 2022; Melin et al. 2021), it is assumed in this study that most new batteries will continue to be produced in the near future in China. When a new NMC 811 battery is displaced, it is therefore expected that the change in production (avoided) will occur in China as they are considered the main marginal supplier of batteries. All data sources for the inventory are listed in Table 1. 4.3 Impact assessment SimaPro 9.3.0.2 (PRé Sustainability 2021) was used to build the inventory model and perform the impact assessment. ReCiPe 2016 Midpoint, Hierarchist (H) perspective, version 1.06 (Huijbregts et al. 2016) is the characterization method used in this analysis. It includes 18 impact categories which are assessed to avoid problem shifting between different environmental areas of protection. Global warming, mineral resource scarcity, and water consumption are the main impact categories highlighted in the “5.3” section as they have been identified as being important in previous battery assessments (Ahmadi et al. 2017). 4.4 Scenario analysis In a market perspective, repurposing batteries can decrease the demand for new batteries and potentially avoid production of these (i.e., avoid impacts from the cradle-to-gate life stage: extraction of raw materials required for new batteries, the processing of the materials, and the battery cell manufacturing). To examine such consequences, three scenarios are assessed based on different substitution coefficients (i.e., degree of decreasing demand of battery due to repurpose of a used LIB). Rigamonti et al. (2020) proposed a procedure based on technical properties for practitioners applying consequential LCA modelling, which is applied in this study. According to the case companies in this study, the remaining battery capacity appears as a crucial technical factor for battery repurposing and is considered as the main Table 1  Data and source Data Source Specification Circular economy business model 1 inventory Circular economy business model 2 inventory Background processes New battery inventory Technical factor determining substitution coefficient Market factor determining substitution coefficient Factors affecting battery market price Case company 1 Case company 2 Ecoinvent v3.8 Ecoinvent v3.8 Both case companies Both case companies Both case companies Resources required for the (foreground) processes Resources required for the (foreground) processes Norwegian electricity mix for remanufacturing and testing Lithium-ion battery with chemistry NMC811 Interview during visit, May 2022 Interview during visit, May 2022 Illustrated in Fig. 2 13 The International Journal of Life Cycle Assessment (2023) 28:554–565 559 factor for determining the technical substitution coefficient for second life LIBs. Battery charge–discharge cycles and calendar aging lead to reduced capacity (measured in kWh) while the second life LIB capacity can also be hindered if the battery was optimized for another application in the first life. Regardless, a battery should have > 60% of the original capacity remaining after end-of-first-life to be used in second life applications (Faessler 2021; Martinez-Laserna et al. 2018). Thus, if an EV battery has aged from 100 to 80% of its original capacity, roughly 50% of the total lifetime is used in its first life and 50% remains for second life. Equation 1 illustrates how to calculate the technical substitution coefficient and exemplifies a 50% capacity remaining. The procedure is based on Rigamonti et al. (2020). TP(SecP) TP(SubP) kWh(SecP) TSC = kWh(SubP) 0.50(SecP) TSC = = 50% 1(SubP) Consumer preference Second life LIB price Valuable materials content Technological development Substitution coefficient Remaining capacity Fig. 2  Key market and technical factor affecting avoided production in battery reuse and repurpose cases. To the left, three business model characteristics affect the dynamic market price TSC = (1) Equation 1 presents the proposed technical property for lithium-ion batteries and example of how the technical substitution coefficient is calculated (TSC, technical substitution coefficient; TP, technical property; SecP, secondary product; SubP, substituted product). Market factors also affect the potential for avoided production as the flow of products and materials are integrated in market structures and mechanisms. A consequence of remanufacturing LIBs and selling these as second life batteries is increased supply of batteries. As a result, the price can be affected through price elasticity of demand. This price effect is present in the circular economy rebound concept by Zink and Geyer (2017) and frames the main market factor affecting the substitution coefficient for this study. If the second life LIB price is lower than a new LIB (often the case currently), the battery may be purchased because of the low price. The three business model characteristics affecting the LIB price illustrated in Fig. 2 were revealed during case company dialogues. Through identifying these characteristics, the assumed LIB price effect on the substitution coefficient becomes more robust. Table 2 shows how the substitution coefficient is identified for the LCA based on the technical and market factors. Equation 2 illustrates the final step to combine the technical and market factor to find the combined coefficient applied in this LCA study. (TC + MC) = CC 2 (0.5 + 0.5) = 0.5 2 (2) Equation 2 presents the final step to calculate the combined coefficient to apply in the LCA (TC, technical coefficient; MC, market coefficient; CC, combined coefficient). 5 Results and discussion 5.1 Life cycle inventory The inventory for CBM 1 represents the required resources to remanufacture and install second life battery packs in a 280 kWh stationary energy storage system. The inventory for CBM 2 represents the required resources to remanufacture and install second life battery modules in a 500 kWh stationary energy storage system. Porzio and Scown (2021) suggest phasing out use of battery mass as functional units in LCAs due to a large variation of chemistries and pack design. Hence, the inventory list in Table 3 is normalized to 1 kWh. 5.2 Life cycle impact assessment The two CBMs are in different substitution scenarios compared with a new battery. The CBMs represent two different second life batteries remanufactured for stationary energy storage. Table 2  How the remaining capacity and the second life battery price affect the substitution coefficient Remaining capacity (%) LIB price Coefficient (%) ≤ 60 80 100 Second life < new Second life < new Second life ≥ new 0 0.5 1 13 560 Table 3  The resources required for circular economy business model (CBM 1 (Eco Stor); CBM 2 (Batteriretur); and a new battery, ready for (second life) stationary storage use per functional unit (1 kWh battery capacity)) The International Journal of Life Cycle Assessment (2023) 28:554–565 Resource Steel rack (reusable one time) 1.250 Battery interface unit 0.005 Cables 0.288 Circuit breakers and junction boxes 0.050 Electricity, Norwegian 1.019 Inverter 0.058 Inverter rack 0.357 Router 0.001 Electricity losses during startup test, Norwegian 0.014 Switch, power supply, e-stop, energy management 0.012 system, extra controller New mini-BMS for modules Copper rail Electronics Main BMS (laptop) Production of NMC811 (Ecoinvent) − 6.7 Table 4 shows the complete life cycle impact assessment (LCIA) results for all 18 categories. The two CBMs are in different substitution scenarios compared with a new battery. The CBMs represent two different second life batteries remanufactured for stationary energy storage. The scenarios with 0% substitution show only impacts from the remanufacturing and installation processes required to transform a used EV battery to a functional second life battery. The scenarios with 50% substitution include negative impacts caused by 50% of the impacts from production of a new battery (− 24.6 * 0.50). The scenarios with 100% substitution include negative impacts caused by 100% of the impacts from production of a new battery (− 24.6 * 1). Thus, the two latter scenario groups show negative impacts in some of the categories. Mineral resource scarcity and terrestrial acidification show negative impacts, also for scenario i. with 0% substitution. This is mainly due to two byproducts from copper processing for the inverter: firstly, the rare-earth metal palladium from electronics scrap in anode slime, and secondly, the rare-earth mineral molybdenite. As impacts associated with by-products are subtracted from the total in consequential LCA methodology, the net impacts can become negative. Smelting of copper concentrate is responsible for the majority of the terrestrial ecotoxicity impacts (553.1 kg 1,4-DCB with 0% substitution). The contribution analysis for the highlighted categories is shown in the supplementary information document. The results show that the difference between the two CBMs is small (≤ 0.1) for all categories. As the CBM 1 case consisted of a 280 kWh sized system and CBM 2 was a 500 kWh system, the impacts from CBM 1 can be marginally lower as the resources required (Table 3) is divided by the total size of the system to obtain the functional unit of 1 13 CBM 1 (pack) CBM 2 (module) New (pack) Unit 0.700 0.288 13.780 0.058 0.008 0.007 1.250 0.005 0.288 0.050 0.058 0.714 0.001 0.014 0.012 kg kg kg kg kWh p kg p kWh kg 0.003 0.083 0.050 0.02 − 6.7 6.7 kg kg kg p kg kWh. Some, e.g., electronics are equal independent of this size range. Figure 3 compares the CBMs with a new battery for stationary energy storage and highlights three impact categories. The results are illustrated with three substitution scenarios (i. 0%, ii. 50%, and iii. 100%). 5.3 Discussion The main results of this study show that the repurposing of used EV batteries for energy storage systems at both the pack and module level is environmentally advantageous compared to using new batteries. This confirms the findings in literature related to battery reuse and repurposing. The three substitution coefficient scenarios in Fig. 3 illustrate the significant effect assumptions on avoided production potential have on impact assessment results. Consequentially, if using the second life battery cannot avoid any production of new batteries, the resulting impacts are higher for choosing one of the battery solutions from the CBMs compared to a new battery. Thus, CBMs utilizing second life LIBs can lower environmental impacts in all categories if some production of a new battery is avoided. This study developed a procedure for assessing the impacts of battery reuse and repurposing and emphasizes the importance of not neglecting substitution coefficients in LCA studies. In scenario iii., the second life batteries from the CBMs fully replace (substitute) a new battery and avoid production and thus environmental impacts, while in scenario i., where 0% substitution is assumed, the second life battery is consumed in addition to a new battery due to market expansion. The consumption of a second life battery will in this scenario not reduce the use of new batteries but increase the total number of batteries on the global market. The International Journal of Life Cycle Assessment (2023) 28:554–565 561 Table 4  LCIA results for CBM 1 (second life battery packs) and CMB 2 (second life battery modules) for all impact categories in three substitution scenarios and a new battery. Values per kilowatt-hour battery capacity CBM 1, 0% CBM 1, 50% CBM 1, substitution substitution 100% substitution CBM 2, 0% CBM 2, 50% CBM 2, substitution substitution 100% substitution New Impact category Unit Global warming Stratospheric ozone depletion Ionizing radiation Ozone formation, human health Fine particulate matter formation Ozone formation, terrestrial ecosystems Terrestrial acidification Freshwater eutrophication Marine eutrophication Terrestrial ecotoxicity Freshwater ecotoxicity Marine ecotoxicity Human carcinogenic toxicity Human noncarcinogenic toxicity Land use Mineral resource scarcity Fossil resource scarcity Water consumption kg CO2 eq kg CFC11 eq 4.04E + 00 − 6.26E + 00 2.76E-06 1.04E-07 − 1.66E + 01 − 2.55E-06 3.97E + 00 − 6.33E + 00 3.03E-06 3.69E-07 − 1.66E + 01 − 2.29E-06 2.46E + 01 8.07E-06 kBq Co-60 eq kg NOx eq 1.91E-01 2.11E-02 − 1.94E + 00 3.96E-02 − 4.08E + 00 5.81E-02 1.91E-01 2.21E-02 − 1.95E + 00 4.07E-02 − 4.08E + 00 5.92E-02 4.46E + 00 − 1.60E-02 kg PM2.5 eq − 4.32E-02 − 3.87E-02 − 3.43E-02 − 4.16E-02 − 3.71E-02 − 3.27E-02 − 5.20E-02 kg NOx eq 2.18E-02 4.01E-02 5.84E-02 2.29E-02 4.12E-02 5.95E-02 − 1.48E-02 kg SO2 eq kg P eq − 1.60E-01 5.22E-03 − 8.86E-02 − 4.25E-03 − 1.76E-02 − 1.37E-02 − 1.53E-01 5.01E-03 − 8.21E-02 − 4.46E-03 − 1.11E-02 − 1.39E-02 − 3.02E-01 2.41E-02 − 4.38E-03 2.46E + 03 − 5.24E + 00 − 5.54E + 00 1.15E + 00 4.70E-03 − 1.28E + 03 9.69E + 00 1.15E + 01 4.63E-01 4.84E + 01 3.63E + 01 8.10E + 01 2.02E + 00 − 3.71E + 00 3.45E + 00 − 7.19E + 00 − 2.36E + 00 6.79E + 00 kg N eq kg 1,4-DCB kg 1,4-DCB kg 1,4-DCB kg 1,4-DCB 1.69E-04 − 2.09E-03 5.53E + 02 1.47E + 03 2.26E + 00 − 1.45E + 00 2.99E + 00 − 1.25E + 00 7.82E-01 9.42E-01 kg 1,4-DCB 5.69E + 01 − 4.36E-03 2.38E + 03 − 5.17E + 00 − 5.48E + 00 1.10E + 00 1.46E-04 − 2.12E-03 6.27E + 02 1.54E + 03 2.19E + 00 − 1.52E + 00 2.93E + 00 − 1.30E + 00 8.27E-01 9.87E-01 4.49E + 01 3.28E + 01 1.95E + 00 − 3.66E + 00 3.38E + 00 − 7.15E + 00 kg oil eq 1.07E + 00 − 2.91E + 00 − 6.88E + 00 1.05E + 00 − 2.93E + 00 − 6.91E + 00 9.02E + 00 m3 5.25E-02 − 3.76E + 00 7.29E-02 − 3.74E + 00 3.86E + 00 m2a crop eq kg Cu eq 5.11E-01 − 1.80E-01 − 1.85E + 00 A lower battery price leads to a higher number of consumers willing to purchase a battery, which means more batteries in total according to the circular economy rebound concept (Zink and Geyer 2017). The remaining battery capacity (technical factor) and battery price (market factor) were identified as important for the substitution coefficient for LCAs assessing reuse and repurpose of batteries. Building on work by Rigamonti and colleagues (2020) and Zink and colleagues (2016), the procedure for calculating the substitution coefficient proposes to account for both factors by combining them in the calculation. Although battery price is recognized as important, it is dynamic and regionally based and thus challenging to use as a parameter in LCA models. To increase predictability, three business model characteristics affecting the LIB price are identified (illustrated in Fig. 2): 1. Demand for second life batteries based on consumer preference (e.g., perception of quality and security). 6.05E + 01 5.85E-01 − 2.24E-01 − 1.83E + 00 2. Valuable materials affect economic value (e.g., older batteries can contain more cobalt). 3. Technological development integrated in new batteries can lower value of the older batteries (e.g., improved battery energy density). These business model characteristics affect the battery price and thus the substitution potential of the second life batteries. By understanding the CBMs assessed, more realistic assumptions were made during the LCA modelling. Hence, CBM insights are valuable when calculating the substitution coefficient to understand the factors affecting the potential for avoided production. By assessing the CBM for batteries proposed in Wrålsen et al. (2021), this study found the potential environmental benefits from this CBM compared to a linear business model. In percentage, the comparison shows that the remanufacturing process of the used batteries corresponds to 16% of the global warming impact of producing a new battery. A 13 562 Fig. 3  Impacts of a second life battery from two different CBMs with no (scenario i.), 50% (ii. dotted line), and 100% (iii. stippled line) avoided production of a new NMC811 battery minimum of 16% production needs to be avoided for second life batteries to contribute to climate change mitigation. For water consumption, this is valid when a used battery can replace 3% of a new battery. For mineral resource scarcity, this is valid even with 0% substitution. CBMs (which aim to gain environmental benefits) should be assessed for different environmental impact categories to quantify potential benefits and avoid problem shifting between different environmental areas of protection. 6 Conclusion This study illustrates the possibilities and advantages of applying life cycle assessment to assess circular economy business models with the case of used lithium-ion batteries 13 The International Journal of Life Cycle Assessment (2023) 28:554–565 from electric vehicles. When comparing the impact of second life batteries facilitated by the circular economy business models with new batteries, results show that the latter has generally higher environmental impacts. Global warming potential results show that the remanufacturing of used batteries corresponded to 16% of emissions from manufacturing. The water consumption corresponded to 3% and mineral resource scarcity to 0%. Despite the relatively low impacts from the battery remanufacturing process, the results indicate that there is a relatively low threshold for environmental benefits by utilizing second life batteries to replace and avoid production of new batteries. For organizations and individuals choosing between new and second life batteries, an investment in the latter is proposed if (1) the remaining technical capacity is sufficient for the new application and (2) the second life battery investment is not performed because of a lower price compared to a new battery but is acquired instead of a new battery. Hence, it should be able to replace a new battery with second life batteries to some extent. If these two technical and market factors are considered, the environmental advantages of the circular economy business models are validated through the results of this study. For LCA practitioners working with consequential modelling, and particularly in circular economy contexts, the substitution coefficient is essential for improving the precision of the life cycle impact assessment results. It is recommended that the potential for avoided production should be identified for each specific study, preferably based on both technical and market factors. The proposed procedure can be applied to other products or product components. Furthermore, business model characteristics affecting market prices can be useful to understand the dynamics and thus improve the substitution coefficient in the model. This study shows that life cycle assessment is valuable to examine if circular economy business models gain net environmental gain as pledged and to evaluate problem shifting to support decision-making. The substitution coefficient appears crucial for impact results, and future research should advance methodology for practitioners to calculate the avoided production potential, also for other second life products. Supplementary Information The online version contains supplementary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 11367-0​ 23-0​ 2154-0. Acknowledgements The authors gratefully acknowledge Mathias Winther Thorsen from Eco Stor AS and Batteriretur who provided the primary data and information from the case second life battery projects. The study is related to the ­2ND LIFE (The value of second life batteries in the future energy system) project funded by the Norwegian Research Council and partners (NFR: 2­ ND LIFE–281005) and the BATMAN project (Lithium ion batteries—Norwegian opportunities within sustainable end-of-life management, reuse, and new material streams) funded by the Norwegian Research Council and partners (NFR: BATMAN—299334). The International Journal of Life Cycle Assessment (2023) 28:554–565 Funding Open access funding provided by University of Agder. Data availability All data generated or analyzed during this study are included in this published article and its supplementary information file. Declarations Conflicts of interests The authors declare no competing interests. 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. References Ahmadi L, Yip A, Fowler M, Young SB, Fraser RA (2014) Environmental feasibility of re-use of electric vehicle batteries. 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Mugerwa, Theophile; Rwabuhungu, Digne Edmond; Ehinola, Olugbenga A.; Uwanyirigira, Janviere; Muyizere, Darius Article Rwanda peat deposits: An alternative to energy sources Energy Reports Provided in Cooperation with: Elsevier Suggested Citation: Mugerwa, Theophile; Rwabuhungu, Digne Edmond; Ehinola, Olugbenga A.; Uwanyirigira, Janviere; Muyizere, Darius (2019) : Rwanda peat deposits: An alternative to energy sources, Energy Reports, ISSN 2352-4847, Elsevier, Amsterdam, Vol. 5, pp. 1151-1155, https://doi.org/10.1016/j.egyr.2019.08.008 This Version is available at: https://hdl.handle.net/10419/243658 Standard-Nutzungsbedingungen: Terms of use: Die Dokumente auf EconStor dürfen zu eigenen wissenschaftlichen Zwecken und zum Privatgebrauch gespeichert und kopiert werden. Documents in EconStor may be saved and copied for your personal and scholarly purposes. 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Ehinola a , Janviere Uwanyirigira b , Darius Muyizere c a Institute of Life and Earth Sciences (Including Health & Agriculture), Pan African University- University of Ibadan, Ibadan, Nigeria School of Mining and Geology, University of Rwanda, Kigali, Rwanda c African Centre of Excellence in Energy for Sustainable Development, University of Rwanda, Rwanda b article info Article history: Received 18 January 2019 Received in revised form 8 July 2019 Accepted 10 August 2019 Available online xxxx Keywords: Peat Proximate analysis Electrical power Energy Rwanda a b s t r a c t In this era, energy and environmental sustainability is a priority in most African countries developmental agenda. The need for energy equity and security in Rwanda is a priority and thus this paper illustrate the current peat resources and their energy potential which can be mined to meet country vast energy requirements. Six hundred (600) peat samples were collected from eighteen peat bogs and all samples were subjected to proximate analyses respecting the procedures outlined by the Bureau of Indian Standard and American Society for Testing and Materials. The high moisture content (average 70.88%) is typical of peats (70%–90%). The total sulfur percentage values (average 0.49%) are generally medium for all samples. The gross calorific values of peat samples ranged from 2560 to 5350 Kcal/kg, with an average value of 3976 Kcal/kg. These values are moderately very high because typical dry peat has calorific values of 2000 Kcal/kg. Mashya displays the lowest value (19%) while the highest ash content (80%) is found in Kageyo. The average ash content in all studied location is 38%. The moisture content in all locations is slightly high ranging from 53% to 85%. Most of the peat deposits in Rwanda may be converted into energy based on the calorific value of all samples. Akanyaru, Mukindo, Gishoma, Mashya, Kaguhu, Mukindo and Nyirabirande are the most promising sites for peat mining. © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1. Introduction Peat is low rank coal, which is made of plant remains as evidenced by the presence of biomarkers detected through chemical, geological, and petrographic studies (Del Rio et al., 1992). It is sedimentary deposit formed principally from plants and rarely from inorganic substances. It is, thus, an aggregate of macerals or premacerals and minerals that when subjected to certain processes, can be converted to coal. According to Cohen et al. (1987) it is difficult to place a precise boundary between what might be a precursor of carbonaceous shale and what might be a precursor of a coal. However, it is agreed that the precursor of a good coal should consist of at least 70% dry weight of organic material (Del Rio et al., 1992). The peat has attracted coal geologists due to their distribution, low ash content, low sulfur content and significant thickness (Staub et al., 1991; Dehmer, 1992). It is used as source of energy mostly in Europe (e.g., Finland, Sweden, etc.,) and Russia (Schora and Punwani, 1980). The use of gas and oil during 20th has shadowed the use of peat (Andriesse, 1988) but the high demand for electricity ∗ Corresponding author at: Institute of Life and Earth Sciences (Including Health & Agriculture), Pan African University- University of Ibadan, Ibadan, Nigeria. E-mail address: luanmugerwa@gmail.com (T. Mugerwa). has stimulated the construction of peat-fired power plant. In developing worlds where energy is scarce, they scale it up to meet their objectives to achieve the development. In Sub-Saharan Africa, energy accessibility and affordability (Equity) is a priority in their agenda while European countries need energy security (IEA, 2014; Ituze et al., 2017). Rwanda is most densely populated in Africa with about 10.5 million inhabitants and it is projected that in 2032, the Rwandan population will be 15.4 million (MINECOFIN, 2014). Inefficient and expensive electrical energy supply hinders the development of the country and therefore the government has a target to decrease wood’s consumption from 86.3% to 50% of national energy consumption in 2020 (Rwanda, 2012). As the demand for energy is growing, the researchers attempted to evaluate the contribution of peat deposits for energy production to meet country vast energy requirements. 2. Peat reserves in Rwanda The quantitative assessment of peat deposits in Rwanda is approximately 155 million tons of peat on dry basis across the country (Vitikka and Lahtinen, 2013; Ekono, 1992) and these deposits could be converted into energy (Hakizimana et al., 2016). The major peat deposits are presented on Table 1. https://doi.org/10.1016/j.egyr.2019.08.008 2352-4847/© 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1152 T. Mugerwa, D.E. Rwabuhungu, O.A. Ehinola et al. / Energy Reports 5 (2019) 1151–1155 Table 1 Peat reserves in Rwanda (EDCL, 2014). Name of peat bog Area (ha) Quantity of exploitable peat reserves, dry basis (tonnes) Sod peat Rucyahabi Akanyaru North–North part Akanyaru North–Middle part Akanyaru North–South part Akanyaru South Mukindo Kaguhu Gishoma Gihitasi Mashya 925 1321 1994 3208 2108 959 195 423 90 36 813 973 501 291 3 572 375 15 740 346 7 797 785 1 323 573 69 712 171 880 12 168 89 821 Table 2 The Von post classification system and Von post degree of humification (H1 to H10) for peat. Source: Adapted from Andriesse (1988). Milled peat Symbol Description 687 998 68 753 2 026 147 11 517 536 6 763 219 698 581 64 942 88 305 12 168 78 191 H1 Completely undecomposed peat which, when squeezed, releases almost clear water. Plant remains easily identifiable. No amorphous material observed present H2 Almost entirely undecomposed peat which, when squeezed, releases clear or yellowish water. Plant remains still easily identifiable. No amorphous material present H3 Very slightly decomposed peat which, when squeezed, releases muddy brown water, but from which no peat passes between the fingers. Plant remains still identifiable, and no amorphous material present H4 Slightly decomposed peat which, when squeezed, releases very muddy dark water. No peat is passed between the fingers but the plant remains are slightly pasty and have lost some of their identifiable features H5 Moderately decomposed peat which, when squeezed, releases very ‘‘muddy’’ water with a very small amount of amorphous granular peat escaping between the fingers. The structure of the plant remains is quite indistinct although it is still possible to recognize certain features. The residue is very pasty H6 Moderately highly decomposed peat with a very indistinct plant structure. When squeezed, about one-third of the peat escapes between the fingers. The residue is very pasty but shows the plant structure more distinctly than before squeezing H7 Highly decomposed peat. Contains a lot of amorphous material with very faintly recognizable plant structure. When squeezed, about one-half of the peat escapes between the fingers. The water, if any is released, is very dark and almost pasty H8 Very highly decomposed peat with a large quantity of amorphous material and very indistinct plant structure. When squeezed, about two-thirds of the peat escapes between the fingers. A small quantity of pasty water may be released. The plant material remaining in the hand consists of residues such as roots and fibers that resist decomposition H9 Practically fully decomposed peat in which there is hardly any recognizable plant structure. When squeezed it is a fairly uniform paste H10 Completely decomposed peat with no discernible plant structure. When squeezed, all the wet peat escapes between the fingers Fig. 1. Main energy resources in Rwanda (RDB, 2019). It is clear that the hydropower energy is the leading energy resources in Rwanda. 3. Energy scenario in Rwanda The main energy resources are Wood, Hydropower, Solar energy, Biogas, wind, biomass, Solar photovoltaic (PV), methane gas, peat, geothermal and petroleum products (Safari, 2010; Ituze et al., 2017). According to RDB (2019) the main energy resources are hydropower; thermal, solar PV, and methane to power (Fig. 1). Rwanda energy sector improvement in the past few years is a result to economic reform, development and clear vision. Wood consumption has been the main source of energy to Rwandan households for the past decades. For example, wood provided 99% of energy in 2000 and 86.5% in 2010, (Rwanda, 2012). Nyamvuba and Gakuba (2014) indicate that supply and consumer needs analysis revealed that 85% of primary energy used in the country were coming from biomass, either in the form of wood directly used as fuel (57%) or charcoal (23%). Bimenyimana et al. (2018) states that, at present day, wood is still the main source of energy, mainly due to its low cost in comparison to other sources. Consequently, however, this large-scale wood consumption has been associated with deforestation, soil degradation (Rwanda, 2012), and have been found to contribute to global warming. In addressing the issue associated with energy production, the Government of Rwanda, in its Vision 2020 policy, planned to have diversified energy resources, provided electricity to 75% of the population and wood consumption to have dropped at 50% of energy consumption, by the year 2020. At present day however, the Rwanda Energy Group (REG) re-drafted targets for electricity generation. REG new planning policy targets on generating 586 MW, from the current 220.9 MW, and providing a 100% electricity access to Rwandans, from the current 45.3%, by the year 2024, (REG, 2019a). This include 52% on-grid and 48% off-grid connections, (USAID, 2018). In addition to wood, Rwanda has various potential resources of energy including biogas, hydro, solar, petroleum, methane gas, wind and geothermal, (Nyamvuba and Gakuba, 2014). Hydropower, solar, and methane gas constitutes the main natural resources of energy in Rwanda (USAID, 2018) (Fig. 1). According to Bimenyimana et al. (2018), hydropower and solar resources are the main renewable energy resources, with the largest power proportion originating from hydropower projects across numerous rivers and water falls across the country. Lake Kivu methane gas production currently contributes ∼14.3% of energy (Fig. 1), and at full completion, the Kivu Watt project aims to produce over 100 MW of electricity (Contour global, 2019). Since 2007, the government of Rwanda has put tremendous efforts in Biogas technology to cut down wood consumption, (Landi et al., 2013). Biogas is mainly used in schools, prisons and some rural households, (Sinaruguliye and Hategekimana, 2013). Currently, Rwanda has not yet discovered any petroleum resources in line with the East African Rift Valley petroleum system. As of 2016, the daily consumption of petroleum products in Rwanda accounted at ∼6000 bbl (US liquid barrel) (Munyaneza et al., 2016). Most of the petroleum products in Rwanda are imported from neighboring countries such as Kenya and Tanzania, (Munyaneza et al., 2016). In addition to the above resources, peat resources are becoming significant contributors to the energy production sector in Rwanda. It accounts for ∼155 million tonnes of dry peat, which can be used for electricity generation (Bimenyimana et al., 2018; REG, 2019b). Seventy seven percent (77%) of such deposits occur T. Mugerwa, D.E. Rwabuhungu, O.A. Ehinola et al. / Energy Reports 5 (2019) 1151–1155 1153 Fig. 2. Rwanda peat deposits distribution map. proximally near Akanyaru and Nyabarongo rivers and Rwabusoro plains (MININFRA, 2015; REG, 2019b). However, despite the large peat deposit accounted in Rwanda, only two peat to power projects are currently under development. Gishoma 15 MW peat to power project was completed in 2017 and 80 MW Hakan project is under construction since February 2017 (REG, 2019b). 4. Materials and methods 4.1. Sampling procedures The systematic grid sampling was used in this study. The sampling process was commenced with determination of the coordinates of all sampling locations and core samples were collected from each drill hole in different locations for each 50 cm interval down to the depth where there is no peat. At each location, the corer was pressed into layers by hand. The six hundred samples collected were placed in tightly closed plastic buckets and labeled prior to being sent to the laboratory for testing. The samples were collected in various peat bogs (Fig. 2). The samples were described on field and classified based on Von Post classification (Table 2). 4.2. Methods The proximate analysis of peat includes the determination of moisture, volatile matter, ash, fixed carbon and calorific value. The first three parameters were determined in laboratory and the fixed carbon was calculated. The samples were crushed to <72-mesh size for chemical analysis. The proximate and sulfur analyses were done following the Bureau of Indian Standard IS:135 (BIS, 2003) and American Society for Testing and Materials (ASTM, 1989) respectively. 5. Proximate analysis results and discussion The average ash content of samples taken from different location are displayed in Table 3 where Mashya display the lowest value (19%) while the highest ash content (80% ) is found in Kageyo (Fig. 3). The average ash content in all studied location is 38%. The moisture content in all locations is slightly high ranging from 53% to 85%. Kaguhu and Kageyo display the lowest moisture content (53%) while Akanyaru North is featured with highest value (85%). The ash content of less than 40% in soda peat dried up to 30% moisture content and less than 30% in milled peat dried to 1154 T. Mugerwa, D.E. Rwabuhungu, O.A. Ehinola et al. / Energy Reports 5 (2019) 1151–1155 Fig. 3. Average ash content in all sampling locations. Table 3 Proximate analysis results of peat samples. Name of peat bog In-situ moisture content(% wt) Average ash content in dry basis Average volatile matter content Average fixed content Average sulfur carbon Average calorific value Cyato Murago Rucyahabi Akanyaru North(other), North Akanyaru North(other), Middle Akanyaru North(other, South) Bishya Akanyaru South (other) Mukindo Gishoma Gihitasi Mashya Kaguhu Bahimba Bisika Kageyo Ndongozi Nyirabirande 64 83 80 68 33 31 29 46 10.70 46.70 55.00 48.13 19.21 20.81 26.40 29.30 0.45 0.12 0.23 0.35 2981 2560 3218 5000.9 78 42 40.83 26.00 0.21 5112 85 28 41 28.10 0.25 4813 70 83 39 20 68.00 60.22 25.70 22.62 0.80 0.41 3654 3782 65 73 73 86 53 55 54 53 76 67 43 31 74 19 28 42 73 80 33 20 60.00 61.36 18.50 50.10 19.98 22.45 31.3 28.88 21.90 19.21 21.00 26.48 25.28 18.22 1.0 0.25 0.53 0.39 0.65 1.1 0.76 0.49 0.64 0.33 4002 4756 2845 4850 5350 3973 3751 2987 3622 4318 40.70 63.90 68.20 72.01 68.21 40% is suitable for electrical power generation (EDCL, 2014). The volatile matter of peat falls between 60%–70% (Lindström, 1980) while that of Rwanda is ranging from 10%–72%. The quality of peats for fuel production is influenced by humification grade and thus all samples with ash content less than 40% are in humification grade 4 (H4) to H10. As demonstrated by Lasse (2007) the increase in humification grade, positively influence the calorific value of peats. The high moisture content (average 70.88%) is typical of peats (70%–90%). The total sulfur percentage values (average 0.49%) are generally medium for all samples. The gross calorific values of peat samples ranged from 2560 to 5350 Kcal/kg, with an average value of 3976 Kcal/kg. These values are moderately very high because typical dry peat has calorific values of 2000 Kcal/kg (Fatma and Sadettin, 2015). The peats with calorific value less than 4562 Kcal/Kg are not suitable energy production (Lasse, 2007). Akanyaru peat bogs have the calorific value above 5000 Kcal/Kg and are considered the overall bogs for energy production. The high ash content of Gihitasi; Bisika and Kageyo make them unsuitable for energy production because the ash content in dry basis above 40% affects negatively the calorific value of peats (EDCL, 2014). According to Ituze et al. (2017), the available peat deposits in Rwanda will contribute 20.6% to the energy growth by 2025 (Table 4). 6. Conclusion The peat to power in Rwanda is a matter of urgency to achieve the developmental target and to ensure that all population will have access to the electricity by 2025. Most of the peat deposits in Rwanda can be converted into energy based the calorific value of all samples. Akanyaru, Mukindo, Gishoma, Mashya, Kaguhu, T. Mugerwa, D.E. Rwabuhungu, O.A. Ehinola et al. / Energy Reports 5 (2019) 1151–1155 Table 4 Rwanda energy mix road map projects (Modified after Ituze et al., 2017). Energy resources Medium term (2012–2017) Medium to long term target from 2018 to 2025 Unit Generation Hydropower Methane Peat power Geothermal Heavy fossil oil Total Average US Dollars (Million) 132–595 200 100 115 160 20 595 510 595–1450 310 350 300 (20.6%) 460 20 1450 555 MW MW MW MW MW MW MW $ Mukindo and Nyirabirande are the promising bogs for peat mining for energy generation. In rural area, the wood is mostly used as sources of power and this has urged the Government of Rwanda and partners to start constructing peat to power plants where the peat are expected to generate 20.6% of all energy resources in Rwanda. This effort to use peat as source of power along with other project, they will promote the development of the Country. To use peat as source of power, it is advisable to develop suitable pollution control as the coal burning contribute to environmental pollution and climate change. The Government is taking initiatives to promote generation of energy from peat including its implications on the environment. Acknowledgments The authors would like to thank African Union Commission through Pan African University for granting the fund to carry out this research. We are thankful to Dr.Nelson Fumo (Editor-inChief) and potential reviewers for their crucial suggestions and comments which have significantly improved the manuscript. References Del Rio, J.C., Gonzalez-Vila, F.J., Martin, F., 1992. Variation in the content and distribution of biomarkers in two closely situated peat and lignite deposits. Organ. Geochem. 18, 67, https://doi.org/10.1016/0146-6380(92)90144-M. Cohen, D.A., Spackman, S., Raymond, J.R., 1987. Interpreting the characteristics of coal seams from chemical, Physical and petrographic studies of peat deposit. Geol. Soc. Spec. Publ. 32, 107–125. http://dx.doi.org/10.1144/GSL.SP.1987.032. 01.08. Staub, J.R., Esterle, J.S., Raymond, A.L., 1991. 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Original Article Incidence and Predictors of Cardiovascular Complications and Death after Vascular Surgery Luciana Andrea Avena Smeili1,2 and Paulo Andrade Lotufo1,2 Hospital Universitário da USP1; Hospital das Clínicas da FMUSP2, São Paulo, SP – Brazil Abstract Background: Patients undergoing arterial vascular surgery are considered at increased risk for post-operative complications. Objective: To assess the incidence and predictors of complications and death, as well as the performance of two models of risk stratification, in vascular surgery. Methods: This study determined the incidence of cardiovascular complications and deaths within 30 days from surgery in adults. Univariate comparison and logistic regression assessed the risk factors associated with the outcomes, and the receiver operating characteristic (ROC) curve assessed the discriminatory capacity of the revised cardiac risk index (RCRI) and vascular study group of New England cardiac risk index (VSG-CRI). Results: 141 patients (mean age, 66 years; 65% men) underwent the following surgeries: carotid (15); lower limbs (65); abdominal aorta (56); and others (5). Cardiovascular complications and death occurred within 30 days in 28 (19.9%) and 20 (14.2%) patients, respectively. The risk predictors were: age, obesity, stroke, poor functional capacity, altered scintigraphy, surgery of the aorta, and troponin change. The scores RCRI and VSG-CRI had area under the curve of 0.635 and 0.639 for early cardiovascular complications, and 0.562 and 0.610 for death in 30 days. Conclusions: In this small and selected group of patients undergoing arterial vascular surgery, the incidence of adverse events was elevated. The risk assessment indices RCRI and VSG-CRI did not perform well for complications within 30 days. (Arq Bras Cardiol. 2015; 105(5):510-518) Keywords: Cardiovascular Diseases/complications; Vascular Complications; Risk Assessment; Postoperative/mortality. Introduction Every year, 250 million major surgeries are performed worldwide, with mortality of 1% and morbidity of 5%. Patients surviving post-operative complications usually have functional limitations and reduced survival 1-4. Patients undergoing vascular surgery are considered at increased risk for post-operative cardiovascular adverse events because: many risk factors that contribute to vascular disease also contribute to coronary artery disease (CAD), such as diabetes and smoking; CAD symptoms can be blurred by low functional capacity; and vascular surgeries can be associated with significant blood volume fluctuations and thrombogenicity 5-7. How to perform a more accurate risk assessment of those patients remains uncertain8-12. Mailing Address: Luciana Andrea Avena Smeili • Hospital Universitário da USP. Rua Aldeia Paracanti, 119 ap. 23, Vila Ré. Postal Code 03667-020. São Paulo, SP – Brazil E-mail: lucianaavena@hu.usp.br Manuscript received March 25, 2015; revised manuscript may 27, 2015; accepted May 28, 2015. DOI: 10.5935/abc.20150113 510 Diseases/surgery; Mortality; Postoperative Two studies (Table 1) have validated the most often used risk assessment models in our practice: revised cardiac risk index (RCRI), derived from a heterogeneous population of patients, of which only a small percentage underwent vascular surgery; and the vascular study group of New England cardiac risk index (VSG-CRI), specific for vascular surgery. However, none of them has approached mortality in their outcomes12,13. This study was aimed at assessing the epidemiological characteristics and clinical outcome of a cohort of patients undergoing vascular surgery, as well as identifying possible predictors of adverse events. Methods This study prospectively assessed a sample of patients admitted to the vascular surgery ward of the Hospital das Clínicas de São Paulo from August 2008 to January 2010. The inclusion criteria were: age ≥ 18 years; patients of both sexes; and elective hospital admission for open vascular surgery or endovascular surgery of occlusive atherosclerotic disease or degenerative aneurysmal disease. The exclusion criteria were: need for emergency surgery; varicose vein stripping; thromboembolectomy; vascular access formation; and refusal to provide written informed consent. Smeili & Lotufo Complications and death after vascular surgery Original Article This study was approved by the Ethics Committee. Statistical analysis The primary objectives were: To assess risk factors, bivariate comparisons of the variables selected were performed in patients with and without the outcomes. Logistic regression was performed, and the odds ratio (OR) and 95% confidence interval for the risk of the outcome in question were calculated. a) to assess the incidence of cardiovascular complications, all-cause death and combined outcome (cardiovascular complications and/or death) within 30 days from surgery; b) to identify possible predictors for those adverse events; c) to analyze whether RCRI and VSG-CRI have good accuracy to estimate the occurrence of cardiovascular complications and total death. Cardiovascular complications were defined as non-fatal myocardial infarction, decompensated heart failure, significant arrhythmia and stroke. The discriminatory capacity of the risk assessment models (RCRI and VSG-CRI) was evaluated by use of the receiver operating characteristic (ROC) curve. The statistical software SPSS, version 17, was used. Results The secondary objectives were: a) to assess the causes of death, classifying them into cardiovascular and non-cardiovascular; b) to assess the incidence of non-cardiovascular complications: wound infection; septic shock; hemorrhagic shock; severe renal failure; respiratory complications; venous thromboembolism; amputation; and reoperation. The patients were classified according to their surgeries as follows: carotid surgery; lower limbs surgery; abdominal aorta surgery; and other surgeries. The following variables were recorded: related to the patient (history, physical exam, laboratory tests, electrocardiography and imaging); related to the surgical procedure; and occurrence of adverse outcomes within 30 days from surgery. Pre-operative clinical variables This study assessed 141 patients, whose mean age was 66 years, 62 patients were 70 years or older, and 92 patients were of the male sex. Table II shows the prevalence of previous diseases. Almost half of the individuals had renal failure, defined as creatinine clearance lower than 60 mL/min, and 19 patients (13%) had creatinine clearance lower than or equal to 40 mL/min. The NT pro-BNP was lower than 100 ng/L in 30%, and greater than 500 ng/L in 30% of the patients. A significant change in the electrocardiogram was observed in 59 (42%) patients, and in the chest radiography, suggestive of heart failure, in 57 (40%) patients. Table 1 – Revised Cardiac Risk Index (RCRI)13 and Vascular Study Group of New England Cardiac Risk Index (VSG-CRI)12 Variable RCRI Points CAD 1 HF 1 Stroke or transient ischemic accident 1 Insulin-dependent diabetes 1 Creatinine ≥ 2.0 1 High-risk surgery: abdominal, thoracic or suprainguinal vascular surgery 1 Variable VSG-CRI Points Age > 80 years 4 Age from 70 to 79 years 3 Age from 60 to 69 years 2 CAD 2 HF 2 COPD 2 Creatinine > 1.8 2 Smoking 1 Insulin-dependent diabetes 1 Long-term beta-blocker use 1 History of angioplasty or coronary revascularization -1 (protective) Points/risk – RCRI: 0 = low; 1-2 = moderate; >2 = high; VSG-CRI: 0-4 = low; 5-6 = moderate; > 6 = high risk. CAD: Coronary artery disease; HF: Heart failure; COPD: Chronic obstructive pulmonary disease. Arq Bras Cardiol. 2015; 105(5):510-518 511 Smeili & Lotufo Complications and death after vascular surgery Original Article Doppler echocardiography was performed in 68% of the patients, 86% of whom had an ejection fraction greater than 50%, while 7% had it lower than 40%. anesthesia (p = 0.010) and of intubation (p = 0.019). Symptomatic peripheral arterial insufficiency correlated inversely with that outcome (p = 0.026); Of the 73% undergoing scintigraphy, almost 40% had a defect, persistent low uptake being observed in 33, transient low uptake in 13, and extensive transient low uptake in 2. – for early combined complication: age (p = 0.044), times of anesthesia (p = 0.000) and of intubation (p = 0.018), use of blood derivatives (p = 0.005) and of vasoactive drugs (p = 0.029), and aorta surgery (p = 0.01). Surgery of the lower limbs (p = 0.003) correlated inversely with that outcome. Regarding perioperative risk assessment, 132 patients (93.6%) were considered ASA II, and 9 (6.4%), ASA III. The RCRI classified 6 patients (4.3%) as at low risk, 63 (44.7%) as at moderate risk, and 72 (51%) as at high risk. According to the VSG-CRI, 34 (24.1%) patients were classified as at low risk, 44 (31.2%) as at moderate risk, and 63 (44.7%) as at high risk. Variables related to surgery The 141 patients underwent the following surgeries: carotid surgery, 15 (106%); lower limbs, 65 (46.1%); abdominal aorta, 56 (39.7%); and other surgeries, 5 (3.5%). Open and endovascular surgeries were performed in 58% and 42% of the patients, respectively. The anesthesia was general or combined in 91.5%. The mean times of anesthesia and orotracheal intubation were 330 min and 875 min, respectively. Blood derivatives were required by 61 patients (43.3%), while vasoactive drugs, by 57 patients (40.4%). Troponin was collected in 128 patients (90.8%), 31 of whom (22%) had it altered. Events in the post-operative period up to 30 days Cardiovascular complications were observed in 28 patients (19.9%), 20 of whom (14.2%) died within that period. Combined complications occurred in 39 patients (27.7%). Early cardiovascular complications were as follows: myocardial infarction, 18 patients; decompensated heart failure, 12; stroke, 3; and arrhythmia, 6. Of the 20 patients dying early, 5 had a cardiovascular cause among the major causes of death (myocardial infarction and/or cardiogenic shock). A non-cardiovascular cause was identified among the major causes of death in 18 patients, being the major cause of death in 15 patients. Septic shock was diagnosed in 10 patients, hemorrhagic shock, in 10, and pulmonary complications, in 4. Thus, only 25% of the deaths had a cardiovascular cause, the non-cardiovascular being the major cause of death in 75%. Non-cardiovascular complications were observed in 55 (39%) patients as follows: pulmonary, 15; renal failure, 15; septic shock, 16; wound infection, 19; venous thromboembolism, 3; and reoperations, 27. Predictors of events The variables with p < 0.05 were as follows: – for early cardiovascular complication: age (p = 0.028), stroke (0.045), obesity (0.025), scintigraphy with transient alteration (0.021), open surgery (p = 0.024), time of anesthesia (p = 0.08), and use of blood derivatives (p = 0.045); – for death: poor functional capacity (p = 0.026), troponina change (p = 0.002), and times of 512 Arq Bras Cardiol. 2015; 105(5):510-518 Assessment of the accuracy of the indices RCRI and VSG‑CRI in the sample The AUC of RCRI and VSG-CRI were, respectively: for early cardiovascular complications, 0.635 and 0.639 (Figure 1); for total mortality, 0.562 and 0.610; and for early combined complication, 0.618 and 0.622. Discussion Clinical characteristics of the sample Table III compares the clinical characteristics found in the studies by Parmar et al14, Bertges et al12, Eagle et al5 and Meltzer et al15 for vascular surgery, and in the study by Lee et al13 for general surgery5,12-15. The following variables were similar in the studies by Parmar et al14, Bertges et al12 and Eagle et al5: age, sex and comorbidities (stroke, chronic obstructive pulmonary disease, diabetes and renal failure)5,12,14. Comparing with the mean of the four studies on vascular surgery, our study showed a higher prevalence of symptomatic peripheral arterial insufficiency, heart failure and arterial hypertension; lower prevalence of previous myocardial revascularization; and high use of acetylsalicylic acid, statin and beta-blocker. More than half of our sample was classified as having poor functional capacity (58%), similarly to the population of the study by Eagle et al5, but higher than that reported by Meltzer et al15. Most of our patients were considered at high or moderate risk according to the RCRI and VSG-CRI scores. We had more endovascular surgeries, a factor that could reduce our risk of events16,17. However, choosing endovascular intervention over open surgery could have been based on a high estimated surgical risk. Another factor that increased the complexity of our sample was the smaller number of carotid surgeries, considered of intermediate risk 10, and predominance of more complex procedures involving the aorta and lower limbs. Our prevalence of ischemia in myocardial scintigraphy was lower than that reported by Eagle et al5. Due to the nature of our patients, elevated prevalence of chronic diseases and clinical evidence of severe ischemia of the lower limbs, in addition to poor functional capacity, we cannot generalize our findings to the general population submitted to vascular surgery. Smeili & Lotufo Complications and death after vascular surgery Original Article Table 2 – Clinical characteristics of the sample < 70 years ≥ 70 years 79 (56.0%) 62 (44.0%) female (%) male (%) 49 (34.8%) 92 (65.2%) absent present Coronary insufficiency 85 (60.3%) 56 (39.7%) HF 64 (45.4%) 77 (54.6%) Dyslipidemia 64 (45.4%) 77 (54.6%) Arterial hypertension 23 (16.3%) 118 (83.7%) Stroke 110 (78.0%) 31 (22.0%) Peripheral arterial insufficiency with claudication 28 (19.9%) 113 (80.1%) Peripheral arterial insufficiency with trophic changes 98 (69.5%) 43 (30.5%) COPD 107 (75.9%) 34 (24.1%) Diabetes 87 (61.7%) 54 (38.3%) Renal failure 73 (51.8%) 68 (48.2%) Smoking habit 30 (21.3%) 111 (78.7%) Revascularization or angioplasty 119 (84.4%) 22 (15.6%) Age Sex Antecedents Low weight 134 (95%) 7 (5%) Overweight 91 (64.6%) 50 (35.5%) Obesity 126 (89.3%) 15 (10.6%) ≥ 4 MET < 4 MET 59 (41.8%) 82 (58.2%) No Yes Statin use 16 (11.3%) 125 (88.7%) Beta-blocker use 44 (31.2%) 97 (68.8%) Acetylsalicylic acid use 14 (9.9%) 127 (90.1%) FC HF: Heart failure; COPD: Chronic obstructive pulmonary disease; FC: Functional capacity. Cardiovascular events and/or death in up to 30 days Total death within 30 days from surgery A high rate of early events was observed, higher than that reported in the literature. In the post-operative period of non-cardiac surgery, the prevalence of total death is low in non-selected populations and in general surgery, ranging from 0.02% to 2.3%. That number, however, is higher when the patient’s or surgery’s complexity increases, reaching 4% for colectomy, 2.9% for revascularization of lower limbs, and as much as 70% for the general surgery of an ASA V patient8,15,18,19. Cardiovascular events in up to 30 days Within 30 days from surgery, the 20% cardiovascular complication rate was much greater than the 2.5% reported by Lee et al13 for general surgery and the 6.3% reported by Bertges et al12 for vascular surgery. That rate was equivalent to the one reported for abdominal aorta open surgery (19.3- 22.6%) by Bertges et al12. The 12% incidence of infarction exceeded the 4.5% reported by Eagle et al 5 , whose sample had a high prevalence of ischemia on scintigraphy, but that study has not used troponin as a diagnostic criterion of infarction. That might have underestimated the number of events found. Regarding total mortality, our 14% exceeded the expected. Previous studies have estimated mortality of 1.5% for carotid surgery, 4.1%-7% for surgery of the lower limbs, and 3.9%-9% for aorta surgery7,20,21. In a large study (6839 patients) with population characteristics similar to ours, mortality within 30 days after amputation of the lower limbs ranged from 9% to 12%22. The total mortality found in this study can reflect the severely-ill sample and its high cardiac complication rate. Arq Bras Cardiol. 2015; 105(5):510-518 513 Smeili & Lotufo Complications and death after vascular surgery Original Article ROC curve 1.0 RCRI VSG-CRI Reference line 0.8 Sensitivity 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 1 - Specificity Figure 1 – Receiver operating characteristic (ROC) curve for the outcome cardiovascular complications. RCRI: Revised cardiac risk Index; VSG-CRI: Vascular study group of New England cardiac risk index. Mortality after post-operative events is high: Gupta et al18 have reported that 61% of the patients developing infarction and cardiac arrest died within 30 days. Similarly, that general mortality can also reflect the high incidence of non-cardiovascular complications (27.7%) in up to 30 days. Meltzer et al15 have reported a high mortality rate for patients with complications: 19% for infectious complications, 33% for cardiac complications, and 42% for pulmonary complications. The outcome ‘cardiac death’ The cardiac death rate was 3.5%, greater that estimated for general surgery in non-selected patients (0.54%-1.8%). Goldman et al 8 have reported that the cardiac death rate can be very elevated, depending on the patient’s clinical characteristics, reaching 56% for those classified as class IV8,18. Outcome ‘cardiac death’ and/or cardiac complication In our study, 33 patients (23.4%) had that outcome. Studies assessing cardiac death and/or cardiac complication in patients with risk factors for ischemic cardiac disease or with known heart disease and general surgery have reported values ranging from 7.9% to 18%9,23. 514 Arq Bras Cardiol. 2015; 105(5):510-518 Non-cardiovascular morbidity in up to 30 days The non-cardiac complication rate found in this study, almost 30%, was higher than the 22% reported by Gawande et al. for colectomy, a surgery known for its high incidence of complications, and the 19% reported by Meltzer et al. for the revascularization surgery of the lower limbs15,19. In 2005, Khuri et al 24 carried out a multicenter prospective study with almost 106,000 patients and reported that post-operative adverse events were more important than pre- and intra-operative variables to determine survival after general surgery. The presence of a complication within the first 30 post-operative days, regardless of the patient’s pre-operative risk, reduced survival by 69%. The presence of pulmonary complication in that study reduced long-term survival by 87%, and the presence of wound complication reduced survival by 42%24. That important finding shows the long-term repercussion of post-operative morbidity. In the presence of cardiovascular complications, one may assume that they will interfere with long-term survival. However, for non-cardiovascular complications, such as wound infection and pulmonary complications, the presence of an exacerbated persistent inflammatory state is suggested to reduce survival24. Smeili & Lotufo Complications and death after vascular surgery Original Article Table 3 – Major predictors of perioperative morbidity and mortality Predictor/ Reference GSH ASA PS 1941 38 Goldman 8 Detsky 9 Eagle 5 Lee 13 Bartel, Reilly, Older Karthikeyan 39 Bertges 12 Parmar 14 Bradbury 34 + + + + Gupta 18 Meltzer 15 31,32,33 + + Age + + + CAD + + + + + HF + + + + + AoS + + Arrhythmias + + Hypoxemia + + Hypercapnia + + Hypokalemia + + Acidosis + + Renal failure + + + + Liver disease + + Bedridden patient + + ITPS + + ITTS + + Aorta surg. + + EMS + + + + + + + + + + + + + Diabetes + + VS + + FC + + Scintigraphy Stroke + + + + + + + COPD + Smoking + BB + Revasc k - SPAIl + + n BNP + + BMI + + GSH: Patient’s general state of health; CAD: Coronary artery disease; HF: Heart failure; AoS: Aortic stenosis; ITPS: Intraperitoneal surgery; ITTS: Intrathoracic surgery; EMS: Emergency surgery; VS: Vascular surgery; FC: Functional capacity; COPD: Chronic obstructive pulmonary disease; BB: Beta-blocker; Revasc.: Myocardial revascularization; SPAI: Symptomatic peripheral arterial insufficiency; BMI: Body mass index; BNP: Brain natriuretic peptide. + Risk factor; – Protective factor. Thus, strategies are required to estimate the risk for non-cardiovascular complications, as well as to monitor and prevent them, because their importance should not be underestimated, despite the smaller number of studies and predictive indices so far available regarding that outcome. Predictors of events within 30 days Regarding the variables related to patients, the following predictors are compatible with literature data: age; stroke; poor functional capacity; and transient low uptake on scintigraphy5,8,12,13,15,18,25-28. The variable obesity requires larger prospective studies to confirm its relationship with that outcome. Differently from our findings, Bradburry et al. have identified lower 2-year survival in low-weight patients with peripheral arterial disease and indication for surgery28. The variable ‘symptomatic peripheral arterial insufficiency’ showed a negative correlation with death within 30 days, differently from that expected15,28 and independently of the type of surgery (open or endovascular). We observed a tendency towards fewer combined events in patients undergoing surgery of the lower limbs and more Arq Bras Cardiol. 2015; 105(5):510-518 515 Smeili & Lotufo Complications and death after vascular surgery Original Article combined events in patients undergoing abdominal aorta surgery. Several previous studies have shown the relationship between surgery of the aorta and the risk for post-operative adverse events8,9,13,18. The lower rate of events found in the group of lower limb surgery might reflect the inclusion of surgeries of heterogeneous complexities (amputation, revascularization, popliteal aneurysm). The following surgery-related factors that correlated with cardiac complications and death reflected the magnitude of surgical stress and tissue injury, independently of underlying diseases and surgery type, although interdependently: time of anesthesia, need for blood derivatives and vasoactive drugs, and time of intubation. Several indices considered surgery magnitude in the risk assessment of post-operative death, such as Possum (1998), the Surgical Risk Scale (SRS - 2002) and the ISIS score (Identification of Risk in Surgical Patients - 2010)28,29,30. The time of anesthesia might have been a predictor and marker of events, reflecting surgery complexity and possible intra-operative events that prolong that time. Studies have shown the importance of intra-operative hemodynamic stability. The Apgar score used three parameters: heart rate, arterial blood pressure and amount of blood loss19. The variables ‘vasoactive drugs’ and ‘blood derivatives’ might have been markers of those hemodynamic changes. As already shown, troponin correlated with death within 30 days. Devereaux et al31 have shown a mortality rate increase within 30 days from 1.9%, for negative troponin in the post‑operative period, to 9.3%, for troponin greater than or equal to 0.30 ng/mL. Accuracy of the risk scores RCRI and VSG-CRI The accuracy of RCRI to discriminate between patients at high or low risk for perioperative cardiac events is considered moderate (AUC of 0.74) for general surgery. However, its performance has been considered low in the subgroup of patients undergoing vascular surgery in the original study (AUC of 0.54 for open surgery of abdominal aorta)13,32. In a systematic review performed by Ford et al32, the performance of RCRI for general surgery was similar to that of the original study (AUC of 0.75); however, for vascular surgery, that index did not show good discriminatory capacity (AUC of 0.64). Similarly, our study showed a good discriminatory capacity of RCRI to predict cardiovascular events in up to 30 days (AUC of 0.635). One limitation of the study by Lee et al13, and also of our study, was to assess the predictive performance of an index only by use of AUC32. VSG-CRI is considered to perform better than RCRI as a predictor of cardiovascular events after vascular surgery (AUC of 0.71). However, it did not show good performance in our study (AUC of 0.639)12. The use of any predictive index requires it to maintain its discriminative capacity in different populations, geographical sites and time. Further studies are necessary to conclude on the value of VSG-CRI among us. 516 Arq Bras Cardiol. 2015; 105(5):510-518 Neither RCRI nor VSG-CRI were good predictors of early death and early combined complications (cardiovascular and/ or death) in our study. More studies are required to test new indices including variables such as age, body mass index, peripheral arterial insufficiency magnitude, functional capacity and myocardial scintigraphy. In addition to a more accurate pre-operative index for clinical risk assessment, another score could be used right after surgery, as has been suggested by Gawande et al. in their proposal to build an Apgar score19. The Apgar score considers higher heart rate and lower arterial blood pressure, in addition to the amount of intraoperative blood loss, showing good correlation with cardiac and non-cardiac complications and death within 30 days19. Based on this study’s findings, we suggest the development of an immediate post-operative score including the following data: times of anesthesia and of intubation, and need for blood derivatives and vasoactive drugs. Because the factors related to patients are not the only responsible for adverse events, a two-time perioperative risk assessment, pre-operative and at the end of surgery, might enhance the accuracy to identify patients in need for greater attention, monitoring and post-operative tests. Conclusions Our sample had a high rate of cardiovascular events and total death in up to 30 days. We observed a greater number of non-cardiovascular deaths than that of cardiovascular deaths, and many non‑cardiovascular complications. Age, stroke, obesity, ischemic alterations on myocardial scintigraphy, aorta surgeries, low functional capacity and troponin changes were identified as risk factors for events in up to 30 days. The risk assessment indices RCRI and VSG-CRI proved not to be good predictors of events. Author contributions Conception and design of the research, Acquisition of data, Analysis and interpretation of the data, Statistical analysis, Obtaining financing and Writing of the manuscript: Lotufo PA; Critical revision of the manuscript for intellectual content: Avena Smeili LAA, Lotufo PA. Potential Conflict of Interest No potential conflict of interest relevant to this article was reported. Sources of Funding There were no external funding sources for this study. Study Association This article is part of the thesis of Doctoral submitted by Luciana Andrea Avena Smeili, from Hospital das Clínicas da Faculdade de Medicina da USP. Smeili & Lotufo Complications and death after vascular surgery Original Article References 1. Weiser TG, Regenbogen SE, Thompson KD, Haynes AB, Lipsitz SR, Berry WR, et al. An estimation of the global volume of surgery: a modeling strategy based on available data. 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W2116717172.txt

Brazilian Journal of Medical and Biological Research (1999) 32: 1063-1071 Photodynamic damage induced in DNA by phycocyanin ISSN 0100-879X 1063 Photodynamic DNA damage induced by phycocyanin and its repair in Saccharomyces cerevisiae M. Pádula1,2 and S. Boiteux1 1Laboratoire de Radiobiologie de l’ADN, Commissariat à l’Energie Atomique, Département de Radiobiologie et Radiopathologie, UMR217 Centre National de la Recherche Scientifique, BP6, Fontenay-aux-Roses, France 2Departamento de Fármacos, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil Abstract Correspondence M. Pádula Departamento de Fármacos Faculdade de Farmácia, UFRJ CCS, Bloco B sub-solo (S 219) 21941-590 Rio de Janeiro, RJ Brasil E-mail: depadula@openlink.com.br Research supported by the Commissariat à l’Energie Atomique (CEA), the Centre National de la Recherche Scientifique (CNRS) and CNPq. Received January 20, 1999 Accepted June 22, 1999 In the present study, we analyzed DNA damage induced by phycocyanin (PHY) in the presence of visible light (VL) using a set of repair endonucleases purified from Escherichia coli. We demonstrated that the profile of DNA damage induced by PHY is clearly different from that induced by molecules that exert deleterious effects on DNA involving solely singlet oxygen as reactive species. Most of PHYinduced lesions are single strand breaks and, to a lesser extent, base oxidized sites, which are recognized by Nth, Nfo and Fpg enzymes. High pressure liquid chromatography coupled to electrochemical detection revealed that PHY photosensitization did not induce 8-oxo7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) at detectable levels. DNA repair after PHY photosensitization was also investigated. Plasmid DNA damaged by PHY photosensitization was used to transform a series of Saccharomyces cerevisiae DNA repair mutants. The results revealed that plasmid survival was greatly reduced in rad14 mutants, while the ogg1 mutation did not modify the plasmid survival when compared to that in the wild type. Furthermore, plasmid survival in the ogg1 rad14 double mutant was not different from that in the rad14 single mutant. The results reported here indicate that lethal lesions induced by PHY plus VL are repaired differently by prokaryotic and eukaryotic cells. Morever, nucleotide excision repair seems to play a major role in the recognition and repair of these lesions in Saccharomyces cerevisiae. Introduction Photosensitizers have been extensively used in DNA repair research due to their ability to induce oxidative damage (1-4). Recently, the relationship among oxidative stress, aging and some degenerative diseases has been demonstrated in humans (5-7). Therefore, photodynamic compounds may constitute an important tool that can be used Key words · · · · Phycocyanin Saccharomyces cerevisiae OGG1 Nucleotide excision repair to mimic oxidative stress and to study the pathways involved in the recognition and repair of these lesions. Phycocyanin (PHY) has been reported to exert a variety of biological effects, including photodynamic action (PDA) (8-10), anti-inflammatory activity in animal models (11,12) and protection against hepatotoxins (13). PHY has also been used as a molecular marker for electrophoretic techniques (14) and as a food additive Braz J Med Biol Res 32(9) 1999 1064 M. Pádula and S. Boiteux (15,16). Its structure is based on an openchain tetrapyrrolic chromophore covalently bound to an apoprotein, which possesses two chromophores attached to the alpha subunit and one to the beta subunit (17,18). Indeed, the photosensitizing properties of PHY are linked to light activation, which seems to be related to the tetrapyrrolic chromophore light absorption, rather than to light absorption by the apoprotein (8,10,19). On the other hand, the anti-inflammatory and scavenging effects of PHY are not related to light activation and may be due, in these cases, to an apoprotein protective action (11,13). If PHY is considered to be a photosensitizer, it may exert its biological effect by two different pathways (type I and type II reactions), with the prevalence of one over the other being dependent on the chemical nature of the sensitizer and on the molecular oxygen content in the reaction (20). In the type I reaction, there is a direct electron transfer from the photosensitizer to the DNA, while in the type II reaction there is an energy transfer or electron transfer from the photosensitizer to molecular oxygen (20). Recently, we demonstrated that DNA damage induced by PHY plus visible light (VL) could be repaired in vitro by Fpg protein and that it depended on the uvrA gene of E. coli in vivo. Furthermore, the photodynamic action of PHY could be inhibited by the addition of atoxic concentrations of sodium azide, indicating the production of singlet oxygen (10). The Fpg, as well as Nth, Nfo and Xth proteins belong to the base excision repair (BER) and the UvrABC complex belongs to the nucleotide excision repair (NER), the most effective pathways that bacterial cells possess to counteract oxidative damage in DNA (21,22). BER and NER are also present in yeast and in human cells (22). Saccharomyces cerevisiae possesses a functional homolog of the bacterial fpg gene, the OGG1 gene, which, like fpg, is responsible for the recognition and repair of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8Braz J Med Biol Res 32(9) 1999 oxodGuo) and 2,6-diamino-4-hydroxy-5-Nmethylformamidopyrimidine (FapyGua) (23). In the present study we used purified DNA repair endonucleases to characterize the chemical profile of PHY-induced lesions in the presence of VL. The identification of DNA damage was based on the substrate specificity of the following DNA repair enzymes from E. coli: the Fpg and Nth proteins for photo-oxidized purines and pyrimidines, respectively, and the Nfo protein for regular and modified abasic sites or AP sites (3,4, 21,24). The production of 8-oxodGuo was monitored in PHY and methylene blue (MB)photodamaged DNA using HPLC coupled to an electrochemical detector (HPLC-ECD). We have also investigated the repair of DNA lesions induced by the PHY and MB photosensitization using the YEplac181 plasmid DNA, which was treated with both photosensitizers in a cell-free system and then used to transform a series of S. cerevisiae DNA repair mutants. The yeast cells were also directly submitted to the photodynamic action of PHY and MB. The results reported here demonstrate that PHY induces photooxidative damages in DNA that represent mostly single strand breaks followed, to a lesser extent, by base-oxidized sites. Moreover, only the NER system of Saccharomyces cerevisiae, but not the OGG1 gene, was found to be involved in the repair of lethal lesions induced by PHY plus VL. Thus, oxidative damage induced by PHY is repaired in different manners in prokaryotic and eukaryotic organisms. Material and Methods Chemicals Methylene blue was purchased from Sigma Chemical Co., St. Louis, MO, USA, and stock solutions (200 µg/ml) were made in PBS and kept at 4oC. Phycocyanin was 1065 Photodynamic damage induced in DNA by phycocyanin purified from Spirulina platensis as previously described (10). Salmon sperm DNA was purchased from Sigma, rehydrated in PBS and kept at -80oC. Proteinase K was purchased from Sigma. and Nfo were prepared from overproducing E. coli strains and purified to apparent homogeneity according to previously described methods and were from our laboratory stock (29-31). Yeast and bacterial strains Photosensitization of yeast and plasmid DNA Escherichia coli JM105 [supE endA sbcB15 hsdR4 rpsL thi D (lac-proAB)/F’ (traD36 lacIq D (lacZ) M15 proA+B+)] was from our laboratory stock in France. All Saccharomyces cerevisiae strains used in the present study and derived from FF18733 [MATa, his7, leu2, lys1, ura3, trp1] were from Dr. Francis Fabre, Institut Curie, Paris. CD138 [ogg1::TRP1], BP10 [rad14::URA3] and BP20 [ogg1::TRP1 rad14::URA3] were from our stock laboratory in France (25,26). Yeast cells were grown in YPD medium and incubated for 24 h at 30oC with shaking. An aliquot was then taken and diluted in fresh YPD medium to obtain a 0.2 A650 nm. This culture was incubated at 30oC with shaking to reach a density of 107 cells/ml. The cells (10 ml) were washed twice with PBS and resuspended in PBS containing PHY (500 µg/ml). The cell suspension was incubated in the dark for 1 h at 30oC with shaking. Irradiation with VL was performed at room temperature in Petri dishes (3.0 x 1.0 cm) containing 2.0 ml of cell suspension as previously described (10). Plasmid YEplac181 (25 µg/ml) was diluted in PBS buffer and incubated in the presence of PHY (500 µg/ml) or MB (2 µg/ml). Each mixture (200 µl) was placed in a 96-well microtiter plate and irradiated at 0oC. Irradiation with VL was performed using a GE PAR 38 “Cool beam” 220 V/150 W lamp as previously described (10). Fluence was measured with a YSI-Kettering model 65A radiometer (Yellow Spring Instruments, Yellow Spring, OH, USA). After PDA treatment, the cell suspension was diluted and plated onto YPD and scored after 48-h incubation at 30oC. MBtreated DNA was ethanol precipitated, washed with 70% ethanol and resuspended in 50 µl of BE16 buffer (26). Phycocyanintreated DNA was incubated with proteinase K (5 µg/ml in BE16 buffer) for 30 min at 37oC and extracted with phenol/chloroform before ethanol precipitation. Cell growth Escherichia coli JM105 was grown in LB broth at 37oC with shaking. The medium was supplemented with ampicillin (100 µg/ ml) when cells were hosting plasmid YEplac181. Yeast strains were grown in YPD medium (1% yeast extract, 1% bacto peptone and 2% glucose) or YNBD minimal medium (0.7% yeast nitrogen base without amino acids and containing 2% glucose) at 30oC with shaking (27). YNBD medium was supplemented with histidine (100 µg/ml), leucine (100 µg/ml), lysine (40 µg/ml), uracil (20 µg/ml) or tryptophan (20 µg/ml) according to the auxotrophic requirements of each yeast strain. Solid medium was prepared by the addition of 2% agar to yeast medium and of 1.5% to the bacterial medium. Plasmid and DNA repair endonucleases Plasmid YEplac181 [LEU2-2µ, AmpR] (28) was prepared from transformed JM105 using the Qiagen midi-prep kit (Qiagen, Paris, France) and stored at -20oC in TE buffer. The DNA repair endonucleases Fpg, Nth Quantification of DNA strand breaks and endonuclease-sensitive sites The standard reaction mixture (20 µl fiBraz J Med Biol Res 32(9) 1999 1066 M. Pádula and S. Boiteux nal volume) contained 0.15 µg of plasmid YEplac181 DNA, either untreated or treated with PHY or MB PDA, and 10 ng of Fpg, Nth or Nfo proteins (4). The reactions were carried out at 37oC for 15 min and stopped by the addition of 3 µl 10% SDS. The reaction mixtures were then submitted to 0.8% gel agarose electrophoresis. The fraction of supercoiled and open circular DNA was determined after ethidium bromide staining and analysis using the Image Store System V.5 and quantified with the NIH Image Program. The average number of strand breaks per circle was calculated assuming a Poisson distribution of the lesions (24). Preparation of competent cells and transformation Transformation of the Saccharomyces cerevisiae strains with YEplac181 plasmid DNA was performed by the lithium acetate method (28). Yeast transformants were plated onto YNBD-agar without leucine and scored after 72-h incubation at 30oC. Detection of 8-oxodGuo in DNA treated with PHY or MB plus visible light The presence of 8-oxodGuo was determined by HPLC-ECD. The analyses were performed using a Waters chromatographic system coupled to an electrochemical detector (Waters, model 460). A C18 µBondapack column equilibrated with methanol/water (5:95, v/v) containing 50 mM sodium citrate, pH 5.0 (BSC solution), was used. The electrochemical detector was calibrated at 700 mV with 100 µl of an 8-oxodGuo standard solution (0.1 µM in BSC). All the analyses were performed under isochratic conditions at 1 ml/min. Salmon sperm DNA (500 µg/ml) was treated with PHY (1 mg/ml) or MB (5 µg/ml) plus VL as described for plasmid DNA in Material and Methods. The samples were ethanol precipitated and resuspended in BE15 (10). A 5-µl aliquot of Braz J Med Biol Res 32(9) 1999 each sample was used to react with Fpg protein (50 µg/ml) for 1 h at 37oC in BE15 (final volume 10 µl). The reactions were stopped by the addition of 90 µl ice cold BSC. The samples were then injected into the HPLC-ECD apparatus. The number of 8oxodGuo/105 DNA bases was calculated by the method of Karahalil et al. (32). Results Enzymatic recognition of DNA lesions induced by PHY In order to determine the chemical nature of DNA lesions induced by PHY plus VL we used a set of DNA repair endonucleases purified from Escherichia coli. Enzymatic recognition of DNA lesions constitutes a strategy that has been widely used for DNA damage characterization. In this test, supercoiled double-stranded plasmid DNA (YEplac181) was treated with PHY or with MB plus VL. After photosensitization, plasmid DNA was incubated with one of the following repair endonucleases from E. coli: Fpg, Nth and Nfo proteins. These enzymes are able to nick DNA at the phosphodiester backbone in a specific manner and induce relaxation of the supercoiled DNA. Single strand breaks corresponded to DNA strand breaks induced by the PDA of PHY or MB without post-incubation in the presence of repair endonucleases. Figure 1 shows SSB and enzyme-sensitive sites in DNA treated with PHY or MB plus VL. The most frequent DNA modifications induced in DNA after PHY photosensitization are single strand breaks, followed by Fpg- and Nth-sensitive sites, which are likely to be damaged purines and pyrimidines, respectively (Figure 1). Nfosensitive sites were less abundant, indicating that base loss sites are produced to a lesser extent after PHY photosensitization. In contrast to PHY plus VL, MB phototreatment induces a large excess of Fpg-sensitive sites and few single strand breaks (Figure 1). 1067 Photodynamic damage induced in DNA by phycocyanin Detection of 8-oxodGuo in DNA treated with PHY and MB plus VL using HPLC-ECD Survival of Saccharomyces cerevisiae cells and of YEplac181 plasmid DNA treated with PHY plus VL None of the yeast strains tested was sensitive to phycocyanin PDA. The FF18733 (wild type), CD138 (ogg1) BP10 (rad14) and BP20 (ogg1 rad14) strains were submitted to PHY plus VL treatment under different conditions: increasing dye concentration, enhanced incubation temperature, increased incubation periods in the dark (over 2 h) and increasing visible light doses. However, even in these conditions, survival of PHY plus VL-treated cells remained the same Sensitive sites/circle In early studies, it was reported that PHY photosensitization may involve the production of reactive oxygen species. The addition of sodium azide protected bacteria against the lethal effects of PHY plus VL (10). Furthermore, the Fpg protein, which recognizes 8-oxodGuo, was able to nick DNA in vitro after PHY plus VL treatment (10). Moreover, 8-oxodGuo is a coding lesion that increases the incidence of GC®TA transversions in bacterial and yeast mutants defective in Fpg and Ogg1 functions, respectively (25,33). These results led us to monitor the formation of 8-oxodGuo in DNA after PHY photosensitization. Using HPLC-ECD, the production of 8-oxodGuo was measured in DNA treated with MB or PHY in the presence of increasing VL doses (Figure 2). In the presence of MB, increasing VL induced 8-oxodGuo in DNA, reaching a plateau at 720 kJ/m2 (1-h light exposure), which may be due to an equilibrium in 8-oxodGuo degradation/formation in DNA (34). On the other hand, after PHY photosensitization, no significant amount of 8-oxodGuo was detected in DNA, indicating that 8-oxodGuo is not produced in DNA or is being produced at a very low rate (Figure 2). 1.0 SSB Nfo Nth Fpg 0.8 0.6 0.4 0.2 0 PHY MB as that of the control cells, without PHY (data not shown). Therefore, we decided to sensitize plasmid DNA and measure its survival after transformation in the S. cerevisiae strains. Figure 3 shows the survival of PHY photodamaged plasmid YEplac181 after transformation into different S. cerevisiae DNA repair mutants. Survival of the PHY photodamaged YEplac181 in ogg1 single mutant was not significantly different from that found in the wild type strain. On the other hand, survival of YEplac181 after PHY photosensitization was greatly reduced in rad14 mutants. The survival of PHY photodamaged YEplac181 in the ogg1 rad14 double mutant was not different from that in the rad14 single mutant. Taken together, these results indicate that the ogg1 mutation does not modify plasmid survival. Therefore, the rad14 mutation, which inactivates NER in S. cerevisiae, seems to play a major role in the elimination of lethal lesions induced by phycocyanin PDA. Figure 1 - Formation of single strand breaks (SSB) or enzymesensitive sites (ESS) in DNA treated with phycocyanin (PHY) or methylene blue (MB) plus visible light. YEplac181 plasmid DNA was either treated with PHY (500 µg/ml plus visible light: 2 min exposure (25 kJ/m2)) or MB (1 µg/ml plus visible light: 12.5-s exposure (2.5 kJ/m2)). Treated or untreated DNA was incubated with 10 ng of the purified E. coli endonucleases: Fpg, Nth or Nfo. The average number of nicks per molecule was determined using 0.8% agarose gel electrophoresis as described in Material and Methods. SSB were induced in DNA treated with MB and PHY plus visible light without incubation in the presence of repair endonucleases. SSB in DNA treated with PHY or MB plus visible light were subtracted from ESS. In all figures, the results obtained correspond to three independent experiments. Survival of Saccharomyces cerevisiae cells and of YEplac181 plasmid DNA treated with methylene blue plus visible light Survival of Saccharomyces cerevisiae was measured after sensitization with MB. Although all strains proved to be sensitive to MB PDA, there were no significant differences in lethality between the wild type strain (FF18733) and the DNA repair mutants (data not shown). Therefore, YEplac181 plasmid Braz J Med Biol Res 32(9) 1999 1068 90 8-oxodGuo/105 DNA bases Figure 2 - Detection of 8-oxodGuo in DNA treated with phycocyanin (PHY) or methylene blue (MB) plus visible light. Salmon sperm DNA was first treated with PHY or MB plus visible light and then with Fpg protein (50 µg/ ml) for 1 h at 37oC as described in Material and Methods. Each final mixture (100 µl) was injected into the HPLC-ECD system. The number of 8-oxodGuo/105 DNA bases was calculated and the limit of sensitivity was six 8-oxodGuo per 105 DNA bases (32). Total exposure time was 1.5 h (1,080 kJ/m2). M. Pádula and S. Boiteux 70 50 MB 30 PHY 10 1.0 Transformation efficiency (%) Figure 3 - Survival of YEplac181 plasmid DNA treated with PHY plus visible light when transformed into wild type (WT) and excision repair mutants of Saccharomyces cerevisiae. YEplac181 DNA was incubated with 500 µg/ml PHY in PBS buffer and irradiated with different doses of visible light. Untreated and treated DNA were used to transform the following competent yeast hosts: FF18733 (wild type), CD138 (ogg1), BP10 (rad14) and BP20 (ogg1 rad14). Total exposure time was 2.0 h (1,440 kJ/m2). WT ogg1 rad14 ogg1 rad14 0.1 0 720 1080 Visible light (kJ/m2) 1440 120 180 Visible light (kJ/m2) 240 1.00 Transformation efficiency (%) Figure 4 - Survival of YEplac181 treated with MB plus visible light when transformed into wild type (WT) and excision repair mutants of Saccharomyces cerevisiae. YEplac181 DNA was incubated with 2 µg/ml MB in PBS buffer and irradiated with visible light. Untreated and treated DNA was used to transform the following competent yeast hosts: FF18733 (wild type), CD138 (ogg1) BP10 (rad14) and BP20 (ogg1 rad14). Total exposure time was 16 min (240 kJ/m2). 360 0.10 WT ogg1 rad14 ogg1 rad14 0.01 0 Braz J Med Biol Res 32(9) 1999 60 80 0 Visible light (kJ/m2) 10 99 0 90 0 81 0 72 0 63 0 54 0 45 36 0 0 27 0 18 0 90 -10 1069 Photodynamic damage induced in DNA by phycocyanin DNA was sensitized to determine the role of BER and NER in the repair of MB plus VLinduced lethal lesions in yeast. Figure 4 shows survival of YEplac181 plasmid DNA treated with MB plus VL after transformation in S. cerevisiae wild type and DNA repair mutants. Similarly to YEplac181 survival with PHY plus VL, when plasmid DNA was sensitized with MB and transformed into S. cerevisiae, its survival was reduced only in the rad14 mutants. The ogg1 mutation had no effect in reducing plasmid survival compared to the wild type strain. Discussion In the present study, we analyzed the damage profile and repair of DNA lesions induced by the photodynamic action of phycocyanin in vitro and in a cellular system. The damage profile of DNA exposed to PHY plus VL was determined using a set of DNA repair endonucleases purified from E. coli. The DNA damage profile of PHY-induced lesions was different from that of MB-induced lesions. While MB plus VL induces a large excess of Fpg-sensitive sites, PHY photosensitization mostly induced single strand breaks (Figure 1). The major difference between these two profiles is in the number of Fpg-sensitive sites generated by the treatments. The excess of Fpg-sensitive sites generated by MB photosensitization can be explained by the production of 8-oxodGuo, which was not detected after PHY plus VL treatment. In fact, the profile of DNA damage induced by phycocyanin was quite similar to that induced by ionizing radiation or by hydrogen peroxide in the presence of transition metals (3,35), but was different from that induced by molecules which involve solely singlet oxygen as the major species responsible for the DNA modifications, such as heat decomposition of 3,3'-(1,4 naphthylidene)-dipropionate endoperoxide, NDPO2 (3). Since PHY photosensitization did not induce 8-oxodGuo in DNA (Figure 2), we conclude that singlet oxygen is not being produced or is immediately quenched by the apoprotein itself. This reasoning is valid when PHY anti-inflammatory properties are associated with oxygen free radical scavenging (12). Since the tetrapyrrolic chromophores are found in the apoprotein core (19), the production and diffusion of singlet oxygen may be hindered by the shielding and quenching effects of the apoprotein. Therefore, PHY photosensitization may occur via a type I reaction. The Fpg-sensitive sites found in DNA after PHY photosensitization may be due to the induction of open imidazole ring purines, such as 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine (MeFapyGua) or 4,6-diamino-5 formamidopyrimidine (FapyAde), which can be found in DNA after reaction with ·OH radical (34). In addition, PHY-photosensitized DNA also presents Nth- and Nfo-sensitive sites, which may constitute oxidized pyrimidine-derived lesions and AP sites, respectively. Phycocyanin was incapable of sensitizing Saccharomyces cerevisiae probably due to a cell wall barrier that prevents phycocyanin from penetrating and exerting its effects. We have shown that phycocyanin could interact with Gram-positive, but not Gramnegative, bacteria, which could justify its effectiveness in sensitizing Staphylococcus epidermidis, but its failure in sensitizing Escherichia coli (10). This same problem arose when MB was used to sensitize DNA repair mutants of E. coli, a fact that led researchers to sensitize plasmid DNA in cell-free systems to study DNA repair (36). For the reasons considered above, YEplac181 plasmid DNA was photosensitized in vitro and used to transform a series of S. cerevisiae DNA repair mutants. Considering the two binomes fpg/OGG1 and uvrA/RAD14, we can propose that the plasmid survival obtained in S. cerevisiae after PHY phototreatment is different from that obtained in bacterial hosts (10). Indeed, after PHY plus VL treatment, plasmid survival was only diminished in the Braz J Med Biol Res 32(9) 1999 1070 M. Pádula and S. Boiteux fpg/uvrA double mutant of E. coli, while its survival in the single mutants was similar to that in the wild type strain (10). Similar results were also obtained when plasmid DNA was treated with MB plus VL and used to transform these same E. coli repair mutants (36). On the other hand, in S. cerevisiae, plasmid survival after PHY photosensitization was greatly reduced only in rad14 mutants (Figure 3). Plasmid survival in the double mutant ogg1 rad14 was the same as that in the single mutant rad14, indicating that an ogg1 mutation against a rad14 background had no effect on plasmid survival. Moreover, plasmid survival in the ogg1 single mutant was similar to that in the wild type strain, showing that the OGG1 gene itself does not play a role in the repair of the lethal lesions induced by PHY plus VL. The same results were obtained for plasmid DNA treated with MB plus VL and used to transform the S. cerevisiae strains (Figure 4). In E. coli, the Fpg protein and NER system are able to recognize PHY- or MB-induced lesions independently and to perform DNA repair. Plasmid survival is only diminished in the absence of both activities, indicating that Fpg and NER recognize the same DNA lesions. In S. cerevisiae, only the NER system is able to recognize PHY- or MB-induced lesions since plasmid survival is not altered by inactivation of the OGG1 gene. Apparently, the difference between bacterial and yeast repair of PHY- and MB-induced lesions may reside in the Fpg/Ogg1 substrate specificity, in a possible BER backup conferred by the Ntg1 protein and, finally, in the NER backup of S. cerevisiae (32,37). If PHY photosensitization does not induce 8-oxodGuo in DNA, the Fpg-sensi- tive sites found after DNA treatment may be due to Me-FapyGua or FapyAde lesions. Both are lethal lesions that can be well recognized by Fpg protein, but only Me-FapyGua is recognized by Ogg1 (21,32). In addition, the Ogg1 protein has a marked preference for AP sites opposite cytosine, while the Fpg protein cleaves AP sites opposite any one of the four DNA bases (32). Although MB produces high levels of 8-oxodGuo, this may not be important in terms of plasmid lethality since it is a pre-mutagenic rather than a lethal lesion (25,38). Besides, if MeFapyGua, FapyAde, and even 8-oxodGuo lesions were not repaired by the Ogg1 protein, they may be recognized by the Ntg1 protein in S. cerevisiae (37,39). Since E. coli does not possess a backup system for oxidized purine lesions, the simultaneous inactivation of the fpg/uvrA will result in plasmid lethality. In S. cerevisiae, since Ntg1 may serve as a backup for Ogg1, plasmid lethality is only observed in the rad14 mutants. Consequently, one may suppose that the NER system of S. cerevisiae differs from that of E. coli in terms of lesion recognition and repair, since the former is responsible for the repair of PHY and MB lethal lesions that are not recognized by Ogg1 or Ntg1. In conclusion, the results reported here demonstrate that PHY photosensitization induces mainly single strand breaks in DNA and, to a lesser extent, base oxidized sites. Furthermore, oxidative damage induced by PHY plus VL is repaired differently in Escherichia coli and in Saccharomyces cerevisiae, a fact that may be due to a BER backup conferred by the Ntg1 protein and to a NER backup of Saccharomyces cerevisiae. References 1. Epe B, Hegler J & Wild D (1989). Singlet oxygen as ultimately reactive species in Salmonella typhimurium damage induced by methylene blue/visible light. Carcinogenesis, 10: 2019-2024. Braz J Med Biol Res 32(9) 1999 2. Menezes S, Capella MAM & Caldas LR (1990). 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ORIGINAL RESEARCH published: 20 June 2022 doi: 10.3389/fpsyg.2022.919254 Function of Perceived Corporate Social Responsibility in Safety of Sports Activities and Home Aerobic Equipment in the Late Period of COVID-19 Lang Ma 1 , Jiang Liu 1*, Yicheng Liu 2 , Yue Zhang 1 and Chunmei Yang 1 1 Physical Education Department, Southwest Jiaotong University, Chengdu, China, 2 Intensive Care Unit, Neijiang Hospital of Traditional Chinese Medicine, Neijiang, China Edited by: Muhammad Irfan, Bahauddin Zakariya University, Pakistan Reviewed by: Rana Tahir Naveed, University of Education Lahore, Pakistan Yang Ying-Fei, Sejong University, South Korea *Correspondence: Jiang Liu liujiang2022w@126.com Specialty section: This article was submitted to Organizational Psychology, a section of the journal Frontiers in Psychology Received: 13 April 2022 Accepted: 20 May 2022 Published: 20 June 2022 Citation: Ma L, Liu J, Liu Y, Zhang Y and Yang C (2022) Function of Perceived Corporate Social Responsibility in Safety of Sports Activities and Home Aerobic Equipment in the Late Period of COVID-19. Front. Psychol. 13:919254. doi: 10.3389/fpsyg.2022.919254 Frontiers in Psychology | www.frontiersin.org The pandemic has impacted various industries, including the sports industry. However, corporate social responsibility (CSR) can mitigate the adverse effects of the crisis and promote the sports industry. To analyze the effect of CSR, the study examined the impact of perceived corporate social responsibility on injury prevention expectation, injury risk perception, and health up-gradation with the mediation of sports safety measures. There are 259 sportsmen of local sports bodies provided the data through a self-administered survey. Data analysis was conducted through Smart-PLS and SEM techniques. The outcome of the analysis showed that perceived corporate social responsibility leads to injury prevention expectation, injury risk perception, and health upgradation. Also, the study found that sports safety measure mediates the relationship between perceived corporate social responsibility and injury prevention expectation, between perceived corporate social responsibility and injury risk perception, and between perceived corporate social responsibility and health up-gradation among sportsmen of local sports bodies. The theoretical implications were presented related to the significance of CSR and sports safety measure and their impact on sportsmen injury prevention expectation, health, and risk perception. The practical implications were related to the management of local sports bodies and how they can induce CSR initiatives and programs. Some limitations related to sample size, incorporating other variables, examining the model in other contexts, and using different study designs, have also been mentioned in the study. Keywords: perceived CSR, health up-gradation, sports safety measures, injury risk perception, injury prevention expectation INTRODUCTION Regular physical activities including sports has not been remain the same during pandemic. Such unexpected changes in activities lead to behavioral modifications of peoples (Dwyer et al., 2020; Woods et al., 2020; Polero et al., 2021). According to Ammar et al. (2020), lockdown during COVID-19 had an impact on public. 1 June 2022 | Volume 13 | Article 919254 Ma et al. Corporate Social Responsibility and Safety of Sports Outdoors sports activities and aerobic activities at home increase respiration rates. While in times of pandemic, it is advised by WHO that those activities which contribute to elevating the respiration rate, must be administered watchfully to avoid any transmission of coronavirus. Therefore, this study tried to explore the risks attached with sports activities, safety measures, injury prevention expectations, injury risk perception and health up-gradation. Moreover, the most important role of perceived corporate social responsibilities (CSR) was also the main aim of the study. For example, immunization has been a greater tool of safety during sports since the invention of COVID-19 vaccines. Sports organizations paid good attention to this aspect of safety of the sportsmen. Insufficient immunization rates were considered as a major public health issue in the developed countries like USA and around the world. Despite the fact that safety measures for sportspersons in amateur sports have received a lot of attention, immunization has been largely ignored in these talks. However, it is considered that sportsmen, viewers and professionals such as referees at recreational sports events are equally at risk of contracting dangerous infections. During early spread of COVID-19, Olympic games to be held in 2020 in Japan were suspended due to the possible spread of disease. It also impacted other professional and amateur sporting events around the world and caused postponement or rescheduling (Francis and Francis, 2020). To overcome these losses, immunization of athletes and participants became a high priority as a safety measure during late times of COVID-19. Sport and recreation participation is crucial for the current and future public health benefits of young population. Psychosocial advantages, increased self-esteem, physical skills development, interpersonal skills, cooperation, competitiveness, reducing stress, and general wellness are all advantages of continued sport engagement in youth. Adult participation rates in sports activities are declining. Sport and physical injury may be contributing to this strain. According to reports, annually 8 % dropouts from sports are due to injury (Emery, 2018). This high rate of injury emphasizes a need for safety measures to lower the risk of injury, prevention from injuries and health up gradation. There is also a need to research the importance of organized team sports in injury epidemiological literature (Emery, 2018). Sports participation may be hampered by minimizing the danger of injury. Sporting activities and sports clubs offer chances and environments for people to be engaged and lead a healthy life. This seems to be a prominent approach considering that physical inactivity is a major health concern right now (Cao et al., 2018). Lower-limb injury accounts for more than 60% of the recreational sports injury burden. While concussion is still a hot topic in youth sports, accounting for 15% of the injury load. Decreased physical activity, increased obesity, posttraumatic arthritis, and post-concussion disorders are all possible consequences of sports injuries (Whittaker et al., 2015). Physical activity involvement is a predictive factor of numerous incidences of deaths (Whittaker et al., 2015). The quality of life can be improved through the encouragement of physical activity and lowering the risk of all sports injury. This is especially required in adventurous and action sports. Injury risk can be investigated For example, reduction in physical activity levels, rise in daily sedentary behavior, and an increase in unhealthy dietary patterns were seen. Some other researchers also got similar results (Oliveira et al., 2021). These sudden developments impacted everyone. The people pursuing their physical activities in gyms, grounds, and other locations were severely impacted. People were compelled to stay at homes because sports facilities and public parks were closed. Closure of such public places disrupted normal sports activities and affected their aerobic workouts. The people were forced to stay at home for a longer period of time. It became difficult for them to maintain their fitness levels. They had to go through the experience of limited physical activities, limited socialization, insecurity, and hopelessness. All these restrictions can contribute to cognitive and emotional health problems (Ammar et al., 2020). Researchers discovered that adults experience psychological problems when they change their existing lifestyles in response to the spread of a disease. However, it is also observed that strong coping skills, abilities, and physical exercises are beneficial in managing such healthrelated issues (Chtourou et al., 2020). People can maintain their fitness through athletics and other sports activities. Such activities are useful in maintaining health status. These also assist in fighting the detrimental consequences of high bp, heart disease, and lung ailments. During the early days of COVID-19 spread, several health-related constraints were seen on rise. This rise was observed majorly due to lack of physical activities. According to researchers, lack of physical activities contributes to shortening of life expectancy and numerous physiological health disorders (Bentlage et al., 2020). Several pulmonary, cardiovascular, neuromuscular, neurological, and skeleton systems are associated with normal body functioning. Sports activities play an important role in regulating these processes. Some of the body processes are associated with maintenance mechanisms (Nawaz et al., 2020b). These include hormone release, gastrointestinal, immunological, and kidney processes. These processes are vital in combating real or fictional risks to the body. All these processes are rectified when individuals get involved in sports activities. Hence, physical exertion and some other precautions are useful in combating the COVID-19 (Chen et al., 2020). According to health experts, regular exercise may reduce the chances of respiratory failure. It has been one of the leading causes of mortality in patients of COVID-19. There is enough evidence suggesting that athletics can contribute to promoting mental well-being (Mazyarkin et al., 2019). It has become extremely hard for some people to achieve the basic criteria of healthy lifestyle given by WHO during COVID-19. The condition is getting worse, as sports and daily workouts are not accessible at these times (Bentlage et al., 2020). There has been a crucial concern of health for people in the wake of restrictions during disease outbreak. They have to survive in these times and seek ways to handle their fitness levels. During home quarantine and locked gymnasiums, playgrounds, and exercise facilities, it is becoming difficult (Hao et al., 2020; Nawaz et al., 2020a). It has been observed that sports activities carry along some of the risks. These risks are associated with sports every time whether on normal days or during pandemic times (King et al., 2020; Lim and Pranata, 2021; Senişik et al., 2021). Frontiers in Psychology | www.frontiersin.org 2 June 2022 | Volume 13 | Article 919254 Ma et al. Corporate Social Responsibility and Safety of Sports systems during late times of COVID-19 which was not studied ever before. after the level of injury in a target group has been determined through surveillance. Following the identification of risk factors, an injury prevention approach is developed and validated. This is done through evaluation of the preventive approach utilizing suitable surveillance (Emery, 2018). In recent years, professional sports have prioritized the operational backdrop for injury prevention. Professional sports organizations are trying their best to provide promising practices and policies (Verhagen et al., 2010). There is a need for cost-benefit analysis while devising policies for injury prevention plans. Such assessments can benefit the process of providing injury prevention measures in sport activities, particularly adventurous sports (Marshall et al., 2016). Currently no studies have gone through injury prevention expectations, injury risk perception and health up gradation of sportsmen in a single framework which showed a significant gap in management research. Moreover, at organizational level, no such resources were provided to the sportsmen with responsibility. Therefore, this research tries to fill the gap by evaluating role of perceived corporate social responsibility on sports safety measures, risk of injury expectations, injury prevention and health up gradation of sportsmen. The global pandemic has a profound impact on public life and sports in a ways that have never been seen previously. The epidemic and lockdowns are an unanticipated external disruption to the sport system. Therefore, sport organizations have to make standard operating procedures every time before proceeding toward tournaments and activities. On one hand, organizations require income from their primary business of sporting events and contests. On the other hand, they also have a financial obligation toward their suppliers and partners (Thormann and Wicker, 2021). The social role of these sports organizations argues that they should act in a socially responsible way. During pandemic, one of the expressions frequently used by sport authorities was “sport’s societal responsibility.” The phrase is defined in policy statements as an essential component of social life that brings people from many cultural origins together. It expresses societal fundamental elements, and promotes public discussion and inclusiveness (Thormann and Wicker, 2021). Corporate social responsibility (CSR) is commonly used to investigate societal responsibilities. The German Football League is a legally recognized organization. This organization applies CSR principles in sports in the very same manner as they do with certain other businesses. Sport regulatory agencies are subject to CSR standards because they are legally organized non-profit organizations that follow the laws of their respective nations (Chelladurai, 2016). Previous research has presented various examples of CSR operation in non-profit sport organizations. The findings emphasized the necessity of CSR in these organizations to project a favorable image, acquire taxpayer support, and improve the friendliness of their primary stakeholders. The pandemic provides a unique opportunity for sport organizations to practice CSR. CSR initiatives may focus on demonstrating overall obligation more than an organization’s financial liabilities (Thormann and Wicker, 2021). Keeping in view the significance of perceived CSR in sports, this research tries to find out the role of CSR in sports safety Frontiers in Psychology | www.frontiersin.org THEORETICAL FRAMEWORK This research gets support from stakeholder theory (Freeman, 1984), which is already in practice by many sports organizations. In order to claim extraordinary standards for themselves, sport organizations rely on significant acceptance from the public (Varmus et al., 2018). Any organization or individual who has a role in achieving the goals of a company is regarded as a stakeholder. There may be some other external stakeholders of sports organizations like supporters and fans. It is difficult to evaluate all these stakeholders through questionnaires. Rather, qualitative approach is suitable and viable to record the viewpoints of them. The variability and size of population suggests that huge number of participants may be evaluated through quantitative approach, and it is useful (Thormann and Wicker, 2021). The stakeholder theory established by (Freeman, 1984) is used in several CSR conceptualizations. Rather than focusing solely on revenue generation for the sake of shareholders, this theory recognizes that firms are accountable and may generate value for a wide range of stakeholders (Freeman, 1984). As a result, some academics emphasized firms’ responsibilities for their customers, employees, authorities, subsequent generations, global ecosystems, and community as a whole (Pérez and Rodríguez del Bosque, 2013). Moreover, one of the primary reasons for sports teams and organizations to embrace CSR practices is pressure from external stakeholders. It highlights the significance of the perception of CSR among stakeholders (Kolyperas et al., 2015). This conceptual basis is consistent with Carroll’s original view of CSR, which is multifaceted in design and incorporates aspects of economic, regulatory, moral, and voluntary duties (Carroll, 1979, 1991). The responsibility of a corporation to produce valuable items for consumers at a reasonable cost, while remaining successful and generating wealth for shareholders, is known as economic stewardship. A regulatory aspect relates to a company’s need to follow the law and adhere to legal provisions in its activities. Moral behavior extends beyond society’s normative expectations. Such behavior provides an adherence to ethical norms even if they are not necessarily governed by legislation. According to Carroll (1979) and Carroll (1991), organizations achieve legitimacy by adhering to ethically correct societal characteristics. Finally, voluntary responsibility encompasses altruistic or charitable activity aimed at the well-being and growth of society. Carroll’s multifaceted CSR conception seems represented in the European Commission’s definition as well. CSR refers to firms having obligations and acting in ways that go beyond their legal requirements and corporate objectives. Such broader obligations encompass a broad range of topics, and they are typically summed up as social and environmental ones. The notion of social refers to community as a whole instead of only social policy matters (Thormann and Wicker, 2021). Scholars have begun to evaluate the emergence of CSR within current strategic policies. These investigations are built 3 June 2022 | Volume 13 | Article 919254 Ma et al. Corporate Social Responsibility and Safety of Sports youth, and their development. Additional actions that can be classified as corporate giving for philanthropic purposes include gifts, ticket freebies, and fundraising campaigns (Inoue et al., 2017; Rowe et al., 2019). This focus on the improvised citizen perception which sees CSR as an instrument for achieving organizational objectives is unusual. Babiak and Wolfe (2006) ended up choosing to probe the morally acceptable and voluntary dimensions of CSR as fulfilled by consensual, implicit actions in their first study. Various factors which encourage organizations to engage in CSR activities were previously investigated. These include external influence from other stakeholders such as competing clubs and internal operations which push professional sports teams’ CSR efforts (Kolyperas et al., 2015). Financial autonomy and human resources are two major motivators for sports federations. The lack of such resources is also one of the most significant barriers to developing formal CSR programs (Zeimers et al., 2021). Researchers have used many techniques to explore CSR in sporting (Lacey and Kennett-Hensel, 2016; Thormann and Wicker, 2021). The results of previous studies used qualitative research methodologies. Qualitative studies were chosen, particularly in professional team sports, with data acquired through observations and interviews, focus groups, and experimental research. The majority of qualitative research was done as case studies of various teams, tournaments, and sports organizations (Babiak and Wolfe, 2006; Banda and Gultresa, 2015; Douvis et al., 2015; Babiak and Kihl, 2018; Walzel et al., 2018). Few investigators utilized a mixed methods approach to address their questions of the study with questionnaires being the most common (Davies and Moyo, 2017). Surveys were used to conduct specialized quantitative research. Mostly, these surveys utilized just brief three to five item measures to assess CSR or perception of CSR, which only scratched the surface of CSR’s multifaceted character (Lacey and KennettHensel, 2016). Walters and Tacon (2013) made an exception by statistically measuring CSR while maintaining the concept’s comprehensiveness. All this literature supported the idea of perceived CSR in sports organizations. Given the popularity of sports activities among sportspersons, most of the existing research on sports-related injuries focuses on elite players or professionals. Physiological changes in both groups, such as muscular strength, physical growth, and variations in combined biomechanics, skill levels, and workloads, the injury landscape, injury risk factors, and injury processes may differ between children and adulthood (Perera et al., 2019). Seemingly, the findings pertaining to grown-up sportspersons cannot always be applied to younger players. Sports participation may be hampered by the danger of injury. Sports and sporting organizations offer chances and environments for people to be active and lead a healthy life, which is especially significant considering that idleness is a major public health issue right now (Cao et al., 2018). Previously, no researcher tried to find out the role of perceived CSR on sports safety measures, injury risk related expectations, injury risk perception and health up-gradation scope of individuals involved in sports activities both outdoors and home-based aerobic activities. The only study conducted on injury prevention expectations, injury risk upon requests for inquiry as to how things work when CSR is introduced in a company (Mirvis and Googins, 2007). Previously, internal and external determinants of social change in an enterprise, as well as the structure of CSR programs and policies have been studied (Russo and Tencati, 2009). However, in sports, a field of CSR which may deal with cooperation, competition and cooperative competition is still developing. Because competitive sport is both a source of direct and indirect competition. This competition may arise between teams and leagues, sports and other forms of entertainment, sponsors and fans. Such competition may be a source of cooperation as well. The institutionalization of CSR may vary across the organizations (Kolyperas et al., 2015). This study also gets support from social exchange theory and social identity theory which focus on performance of sportsmen (Back, 1965). If they are given socially responsible safety measures during the course of sports activities, then in exchange they may perform better in sports. So, the provision of safety measures during sports activities works exchange-ably with the efforts put in by the sports organizations or teams. Similarly, drawing upon social identity theory, CSR positively predicts the performance of the players, or the ones involved in sports activity (Askarian and Raahbar, 2021). Injury prevention expectation, injury risk perception and health up gradation are all possible outcomes of positive outcomes of perception of CSR in directing the safety measures in sports activities. Therefore, this study is strongly supported by stakeholders’ theory, social exchange and social identity theory. Perceived CSR, Sports Safety Measures, Injury Prevention, Injury Perception and Health Sport organizations have historically been known for their capacity to deliver social benefits such as better physical health, improved education, and social integration (Jarvie, 2003; Myers et al., 2004; Lambourne, 2006). The idea of CSR has exploded in popularity all across major sports sectors. It’s becoming more common in professional sports, including an expanding list forming partnerships, economic, and societal entities in order to effectively engage with a variety of stakeholders. CSR perception in sporting settings, particularly clubs, is important for managers and sports management staff. Because sport companies differ from one another and from other enterprises of main activities, organizational structures, mindsets, and strategies (Dar et al., 2022). It is important to understand how CSR activity unfolds, develops, and evolves within such unusual and socially created organizations. It is also useful to explore causes, obstacles, and organizational stages to develop additional CSR perceptions about athletics as well as other domains of sports (Kolyperas et al., 2015). Several studies on sport organizations’ CSR have been undertaken in a variety of situations such as professional team sports, sport competitions, and sport federations (Babiak and Wolfe, 2006; Walters and Tacon, 2013; Walzel et al., 2018). Prior study on CSR activities focused on community-based activities with an emphasis on sports activities, education of Frontiers in Psychology | www.frontiersin.org 4 June 2022 | Volume 13 | Article 919254 Ma et al. Corporate Social Responsibility and Safety of Sports and analyze the data on the injury crisis, and then distributing the results to organizations with the means and authority to take preventive action (Norris and Pittman, 2000). There are a number of factors to consider when addressing sports safety management from a community standpoint. Some athletes, for instance, are members of groups that transcend geographical bounds. They form sports societies which can concentrate on participation in a regional sports organization (Timpka et al., 2006). If contemporary society health and safety promotion strategies are to be shown in the wide variety of sports, program designs must include making allies with all these global and local sports community members at all tiers. These strategies work well by collecting and processing detailed information through the findings for strategic planning in the pertinent organizational setting. Nonetheless, sporting organizations alone are unable to address all concerns relating to sports safety such as challenges with inadequate sporting venues and drug and alcohol addiction. In order to build safe local services and settings for physical activities, a community-based sport injury prevention program must form partnerships with geographically specified groups (Ross et al., 2021). Currently, no study has found any significant contribution of sports safety measures due to a lack of research. Although, this has a lot of scope to be explored. Therefore, this study tries to find out the mediating as well as direct roles of sports activities and injuries in terms of prevention expectations, injury perception and health upgradation of people during the late pandemic of COVID-19. This study utilized outdoor sporting activities and home-based aerobic physical activities to be monitored. The above literature in support of sports safety measures also indicated that the proposed role of sports safety measures can mediate between perceived CSR, injury prevention expectation, injury perception and health up-gradation. Therefore, the author tried to find out the possible associations between them through these hypothesis. perception and problems related to health, was in the field of floorball sport (Perera et al., 2019). This research motivated the authors to conduct the current research. It suggested the possible associations between perceived CSR and these dimensions of injury panorama. Therefore, the following were suggested. H1 : Perceived corporate prevention expectation H2 : Perceived corporate safety measures H3 : Perceived corporate risk perception H4 : Perceived corporate up gradation social responsibility leads to injury social responsibility leads to sports social responsibility leads to injury social responsibility leads to health Sports Safety Measures Within the sports management and public health areas, the debate over what duties sports organizations have toward society is gaining traction. Internationally, sports organizations’ social responsibility in relation to health improvement strategies and practices has been explored (Kokko et al., 2014). However, little research has been done on how managerial ideals of social responsibility and health up-graduation might be integrated inside sports groups. Participation in group sport adds significantly to recreational physical exercise at health-up gradation levels. Community sports clubs are crucial venues for overall physical activity. Sport is a widely used form of recreational physical activity, particularly amongst children and teens. Additionally, involvement in group sports can have a significant impact on not only physiological but also mental and social health. Because of the communal element of group sports, the social and mental health advantages can be greater than those obtained from participating in individual types of sports activity. Setting-based health up gradation emphasizes pretty much on the entire thinking to handle a variety of behavioral change tactics which promote sports engagement as a healthy habit. It can lead to encouraging people to participate in sports. Interpersonal level elements such as enhancing competence and ability, intrapersonal techniques such as better coaching practices, and physical environmental and policy level effects such as boosting organizational governance and management are all examples of these strategies (Kokko et al., 2014). In sport organizations around the world, a variety of health promotion methods and policies are being applied. Sport injury prevention, cigarette free locations, appropriate alcohol consumption, sunscreen, eating healthy, drinking healthy beverages, and friendly environments are just a few of them (Kokko et al., 2016). Some are more focused on governmental level. Such strategies and tactics are sometimes led by the organizations themselves or by external health promotion organizations. Although the impact of environmental framework depicts sports injury mechanisms and connects these to the effects and environmental intervention areas. It lacks precise recommendations for developing and implementing practical safety promotional strategies. Contemporary health and safety promotion strategies are built on community mobilization by gathering together relevant agencies and specialists to gather Frontiers in Psychology | www.frontiersin.org H5 : Sports safety measures lead to injury prevention expectation H6 : Sports safety measures lead to injury risk perception H7 : Sports safety measures lead to health up gradation H8 : Sports safety measures mediate the relationship of perceived CSR and injury prevention expectation H9 : Sports safety measures mediate the relationship of perceived CSR and injury risk perception H10 : Sports safety measures mediate the relationship of perceived CSR and health up gradation The theoretical framework devised from the literature review has been given in Figure 1. METHODOLOGY The study intends to examine the impact of perceived corporate social responsibility on injury prevention expectation, injury risk perception, and health up-gradation. The study also analyzed the mediating effect of sports safety measures. The proposed hypotheses of the study have been developed based on the research objectives, and these hypotheses were analyzed through 5 June 2022 | Volume 13 | Article 919254 Ma et al. Corporate Social Responsibility and Safety of Sports FIGURE 1 | Theoretical framework. PCSR, Perceived Corporate Social Responsibility; IPE, Injury Prevention Expectation; IRP, Injury Risk Perception; HU, Health Up Gradation; SSM, Sports Safety Measures. Smart-PLS 3.3.3 opted for data analysis. Cho et al. (2020) claimed that Smart-PLS uses path models (such as measurement models and structural models) to analyze the data quickly. The models test the significance of research hypotheses along with data validity and reliability. The models also help the research to either reject or accept the proposed hypotheses by analyzing t-statistics and p-values (Avotra et al., 2021). a deductive approach. The reliability of the data was ensured using a quantitative research design and this research design reduced any biases in the data. The data collection procedure was carried out using a self-administered survey. This survey has clarity and precision for maintaining the rationality of the data. To carry out the study, a total of 350 questionnaires were disseminated among the participants of the study. Within a time of 3 weeks, a total of 259 questionnaires were completed and properly filled out by the participants. The rest of the questionnaires were discarded during the screening procedure. After the data was collected, the data was analyzed with the aid of suitable statistical software. The target population was sportsmen of local sports bodies of China. A non-probability convenience sampling strategy was opted to select the sample from the whole target population. Nawaz et al. (2022) claimed that this method of selecting the sample takes convenient and efficient as the data is obtained from the readily available respondents. The data was obtained from individuals (unit of analysis) as the target population was sportsmen of local sports bodies of China. Demographics Details The demographic profile of the study participants has been presented in Table 1 below. These demographic factors of gender, age and education have been incorporated in the survey to get an idea about the educational background and degree of participation of each gender. Since these factors are considered important and fundamental to the sports individuals. The male and female sports persons participated in the study with 64.48% male participation and 35.52% female participation. This also showed a difference in the gender participation endorsing the findings of previous research (Burton, 2015). Age of 15 years and above is added because according the sporting rule and body for sports (in alignment with Berlin 5th Consensus on Concussion in Sport) shows that risk of injury is reduced by 60% in ages above 13 (Emery, 2018). Further, the participant taking part in the sports even formally as player (not trainer) were above age 15.The sportsmen aged between 15 to 20 years were 154 while 105 sportsmen were between 21 and 30 years. Moreover, most of the participants had bachelor’s degree (65.64%), whereas master’s degree holders and Ph.D. and other degree holders were 25.87 and 8.49%, respectively. Measurement The survey contains a 5-Point Likert scale which aided in obtaining the data from the study participants. The sections below described the measurement scale for each construct of the study. The measurement scale of perceived corporate social responsibility had 8-items which were adopted from Zhang et al. (2021). The measurement scale of injury prevention expectation had 2-items which were adopted from Emery (2018). The measurement scale of injury risk perception had 3-items which were adopted from Emery (2018). The measurement scale of health up-gradation had 5-items which were adopted from Emery (2018). The measurement scale of sports safety measures had 9-items which were adopted from Zhang et al. (2021). The structured Equation Modeling (SEM) technique was deployed to analyze the relationship between the study variables. As the study used this technique, the most suitable software Frontiers in Psychology | www.frontiersin.org DATA ANALYSIS AND RESULTS Measurement Model The output of the measurement model which presents the relationship between the independents variables with dependent variables can be seen in Figure 2. 6 June 2022 | Volume 13 | Article 919254 Ma et al. Corporate Social Responsibility and Safety of Sports The model assessment (direct model) is demonstrated in Table 2. The factor loadings for PCSR are between 0.728 and 0.787, for IPE are between 0.879 and 0.908, for IRP are between 0.887 and 0.894, for HU are between 0.818 and 0.915, and for SSM the factor loadings are between 0.786 and 0.856. The values obtained are above the 0.60 threshold level; therefore, all the values lie in the acceptable value range (Shah et al., 2021; Yingfei et al., 2021). Moreover, the obtained values of VIF are below 5 which the highest VIF value of 4.825, which indicates that there is no issue of collinearity in the data (Hair et al., 2017). The construct reliability and validity using Cronbach alpha values, composite reliability, and AVE are also shown in Table 2. The results showed that internal consistency exists between the items of the variables as the values obtained are higher than 0.70 as PCSR (α = 0.941), IPE (α = 0.748), IRP (α = 0.844), HU (α = 0.923), SMM (α = 0.941) (Gualano et al., 2011). The data reliability was also analyzed using composite reliability and its value must be 0.70 (Peterson and Kim, 2013). The values obtained for composite reliability were above the acceptable value, thus the data was said to be reliable. Convergent validity was examined using AVE values and AVE should be higher than 0.50 (Archer et al., 2021). AVE values for the present study are above this threshold level (AVE = 0.573-AVE = 0.798). Therefore, indicating the presence of convergent validity. Discriminant validity using two tests (i.e., Fornell-Larker Criterion and Heterotrait-Monotrait (HTMT) ratio) have been demonstrated in Table 3 below. The result obtained for FornellLarker Criterion shows that the value at the top of the column is greater than the following values on the same column (Fornell and Larcker, 1981). The discriminant validity test based on TABLE 1 | Demographics analysis. Demographics Frequency Percentage Gender Male 167 64.48% Female 92 35.52% 15–20 154 59.46% 21–30 105 40.54% Age (years) Education Bachelors 170 65.64% Masters 67 25.87% Ph.D. and others 22 8.49% N = 259. FIGURE 2 | Output of measurement model. PCSR, Perceived Corporate Social Responsibility; IPE, Injury Prevention Expectation; IRP, Injury Risk Perception; HU, Health Up Gradation; SSM, Sports Safety Measures. Frontiers in Psychology | www.frontiersin.org 7 June 2022 | Volume 13 | Article 919254 Ma et al. Corporate Social Responsibility and Safety of Sports TABLE 2 | Model assessment (Direct Model). Construct reliability and validity Factor loadings VIF PCSR1 0.751 1.880 PCSR2 0.774 2.538 PCSR3 0.734 2.178 PCSR4 0.728 1.720 PCSR5 0.787 2.996 PCSR6 0.764 3.220 PCSR7 0.738 2.793 PCSR8 0.775 4.791 IPE1 0.908 1.557 Injury prevention expectation IPE2 0.879 1.557 Injury risk perception IRP1 0.883 2.075 IRP2 0.887 2.217 IRP3 0.849 1.858 HU1 0.882 4.489 HU2 0.892 3.637 HU3 0.915 4.169 HU4 0.818 2.556 HU5 0.866 3.131 SSM1 0.786 2.944 SSM2 0.840 3.899 SSM3 0.856 4.102 SSM4 0.812 4.229 SSM5 0.789 2.561 SSM6 0.811 3.136 SSM7 0.855 3.929 SSM8 0.814 4.825 SSM9 0.855 4.053 Perceived corporate social responsibility Health up gradation Sports safety measures α Composite reliability AVE 0.895 0.915 0.573 0.748 0.888 0.798 0.844 0.906 0.763 0.923 0.942 0.766 0.921 0.930 0.680 PCSR, Perceived Corporate Social Responsibility; IPE, Injury Prevention Expectation; IRP, Injury Risk Perception; HU, Health Up Gradation; SSM, Sports Safety Measures; VIF, Variance Inflation Factor; α, Cronbach Alpha; AVE, Average Variance Extracted. TABLE 3 | Discriminant validity. Fornell–Larcker criterion Constructs HU IPE IRP Heterotrait–Monotrait ratio PCSR HU 0.875 IPE 0.592 0.894 IRP 0.626 0.627 0.873 PCSR 0.543 0.596 0.554 0.757 SSM 0.568 0.467 0.778 0.484 SSM Constructs HU IPE IRP PCSR SSM HU 0.825 IPE 0.711 IRP 0.710 0.786 PCSR 0.581 0.702 0.608 SSM 0.599 0.543 0.864 0.496 N, 259; PCSR, Perceived Corporate Social Responsibility; IPE, Injury Prevention Expectation; IRP, Injury Risk Perception; HU, Health Up Gradation; SSM, Sports Safety Measures. shows the values of HTMT ratio and the values are below 0.90 (HTMT = 0.864-HTMT = 0.496), thus discriminant validity based on HTMT ratio exists (Benitez et al., 2020). R-square values for the variable of the study can be viewed in Table 4. Hair et al. (2014) claimed that the value of R-square near 0.50 indicates that the model is sustainable. The result obtained Fornell-Larker Criterion indicates that discriminant validity exists (Afthanorhan et al., 2021). The table shows that the top value of each column for heath upgradation is 0.875 and the rest of the values in the same column are lower than this. The top column value for IPE is 0.894 and the rest of the values in the same column is lower than 0.894. Table 3 also Frontiers in Psychology | www.frontiersin.org 8 June 2022 | Volume 13 | Article 919254 Ma et al. Corporate Social Responsibility and Safety of Sports leads to health upgradation. The relationship between PCSR and HU is weak (f2 = 0.161). H5 also got accepted as (t = 3.139; p < 0.05) which means that sports safety measure leads to injury prevention expectation. SSM has a very weak relationship with IPE (f2 = 0.069). H6 was accepted (t = 11.214; p < 0.05) which means that sports safety measure leads to injury risk perception. SMM is very strongly related to IRP (f2 = 0.959). Moreover, H7 proposed that sports safety measure leads to health upgradation, and this hypothesis was accepted (t = 5.672; p < 0.05). The relationship between SSM and HU is weak (f2 = 0.209). The results for the indirect effects (H8 to H10) are demonstrated in Table 6. H8 got accepted as (t = 3.059; p < 0.05) which means that sports safety measure mediates the relationship between perceived corporate social responsibility and injury prevention expectation. The result for H9 (t = 7.516; p < 0.05) indicates the acceptance of the hypotheses, which signifies that the sports safety measure mediates the relationship between perceived corporate social responsibility and injury risk perception. H10 also got accepted as (t = 5.242; p < 0.05) which means that sports safety measure mediates the relationship between perceived corporate social responsibility and health upgradation. TABLE 4 | R-Square values for the variables. R-Square HU 0.412 IPE 0.392 IRP 0.643 SSM 0.232 N, 259; PCSR, Perceived Corporate Social Responsibility; IPE, Injury Prevention Expectation; IRP, Injury Risk Perception; HU, Health Up Gradation; SSM, Sports Safety Measures. for R-square for health upgradation is (R2 = 0.412), for injury prevention expectation is (R2 = 0.392), for injury risk perception is (R2 = 0.643), and for sports safety measures is (R2 = 0.232). According to Craney and Surles (2007), inner VIF values should be below 5 to claim that there is no issue of collinearity in the data. The results of inner VIF values explain the collinearity statistics of the data and the results for this index show that the inner VIF for HU and PCSR is (VIF = 1.306), for HU and SSM is (VIF = 1.306), for IPE and PCSR is (VIF = 1.306), for IPE and SSM is (VIF = 1.306), for IRP and PCSR is (VIF = 1.306), for IRP and SMM is (VIF = 1.306), for SMM and PCSR is (VIF = 1.000). As all the values are below 5, thus no collinearity issue was detected in the data set. DISCUSSION Structural Model This research tries to find out the possible associations between different organizational features of sports and sport-related matters during the late era of COVID-19.This research tries to find out direct as well as indirect associations which aid in boosting relationships. The duration of the pandemic affected the people in many ways which influenced their daily habits and routines. The people were confined to their homes and physical activities got limited due to the spread of viruses. This all continued for almost 2 years. Sports activities started to revamp in the late era of this pandemic and bear along with some cautions and preventive measures. As with many physical activities, there arose a concern about safety in sports activities as well. Although sports safety was already in consideration with a number of sports activities, but this pandemic put more strain on the sports activities when it came to the spread of the virus. Most of the countries restarted their sports activities with certain preventive measures in practice such as distancing, sanitization, protective equipment and secure bubbles (Woods et al., 2020). This research contributes significantly to the sports literature as it tries to find the direct relationships of perceived CSR with sports safety measures, injury prevention expectations, injury risk perceptions and health up-gradation of people. The direct effects showed that perceived CSR was significantly associated with sports safety measures and this injury related panorama. These results indicated that when organizations connected with sports activities at outdoors and indoors and even with the sports equipment manufacturing are managed responsibly keeping in view the safety of people The value of t-statistics has been shown in Figure 3 below. This figure demonstrates the output of structural model bootstrapping. A 95% confidence interval has been taken to accept or reject the hypotheses of the study. The results for the direct and indirect effects have been shown in Tables 5 and 6, respectively. The values of t-statistics and p-values determine the acceptance or rejection of the study hypotheses. A value of more than 1.96 for t-statistics signifies the acceptance of the hypotheses (Winship and Zhuo, 2020). Whereas, a value of < 0.05 or with a 95% confidence interval for p-value suggests the acceptance of the study hypotheses (Pavlov et al., 2020). The effect size using the f-square value is also shown in Table 6. According to Cohen (1992), the value of f-square closer to 0 means the effect is weak, while, the value near to 1 means the effect is strong. The results for H1 to H7 (direct effects) can be seen in Table 5. The result for H1 (t = 7.155; p < 0.05) indicates the acceptance of the hypotheses, which signifies that the perceived corporate social responsibility leads to injury prevention expectations. The relationship between PCSR and IPE is weak (f2 = 0.296). H2 also got accepted as (t = 8.848; p < 0.05) which means that perceived corporate social responsibility leads to sports safety measures. PCSR has a weak relationship with SSM (f2 = 0.306). The outcome of H3 (t = 3.723; p < 0.05) shows that the hypothesis is accepted which means that perceived social corporate responsibility leads to injury risk perception. PCSR has a weak relationship with IRP (f2 = 0.115). The result for H4 (t = 4.858; p < 0.05) indicates the acceptance of the hypotheses, which signifies that the perceived corporate social responsibility Frontiers in Psychology | www.frontiersin.org 9 June 2022 | Volume 13 | Article 919254 Ma et al. Corporate Social Responsibility and Safety of Sports FIGURE 3 | Output of structural model bootstrapping. TABLE 5 | Direct effects of the variable. Paths H O M SD T-statistics Effect Size (f 2 ) P-value Results PCSR→IPE H1 0.483 0.492 0.067 7.155 0.296 0.000*** Accepted PCSR→SSM H2 0.484 0.487 0.055 8.848 0.306 0.000*** Accepted PCSR→IRP H3 0.231 0.235 0.062 3.723 0.115 0.000*** Accepted PCSR→HU H4 0.350 0.355 0.072 4.858 0.161 0.000*** Accepted SSM→IPE H5 0.233 0.225 0.074 3.139 0.069 0.002** Accepted SSM→IRP H6 0.666 0.663 0.059 11.214 0.959 0.000*** Accepted SSM→HU H7 0.399 0.393 0.070 5.672 0.209 0.000*** Accepted N = 259; p*** < 0.001, p** <0.005, p* <0.05, H, Hypothesis; O, Original Sample; M, Sample Mean; SD, Standard Deviation; SRMR= 0.096, NFI=0.669, PCSR, Perceived Corporate Social Responsibility; IPE, Injury Prevention Expectation; IRP, Injury Risk Perception; HU, Health Up Gradation; SSM, Sports Safety Measures. TABLE 6 | Indirect effects of the variable. Paths H O M SD t-statistics P-value Results PCSR→ SSM→ IPE H8 0.113 0.109 0.037 PCSR→ SSM→ IRP H9 0.322 0.322 0.043 3.059 0.002** Accepted 7.516 0.000*** PCSR→ SSM→ HU H10 0.193 0.190 0.037 Accepted 5.242 0.000*** Accepted N = 259, p*** <0.001, p** <0.005, p* <0.05, H, Hypothesis; O, Original Sample; M, Sample Mean; SD, Standard Deviation; PCSR, Perceived Corporate Social Responsibility; IPE, Injury Prevention Expectation; IRP, Injury Risk Perception; HU, Health Up Gradation; SSM, Sports Safety Measures. Frontiers in Psychology | www.frontiersin.org 10 June 2022 | Volume 13 | Article 919254 Ma et al. Corporate Social Responsibility and Safety of Sports THEORETICAL IMPLICATIONS, PRACTICAL IMPLICATIONS, AND CONCLUSION then it could lead to practices which help in people’s wellbeing. When the organizations are responsibly managed in terms of social safety and environmental safety then it leads to devise good protocols which aid in managing the safety of the participants whether they are players or spectators. This can be better understood with the phenomenon of CSR in sports organizations which has been well studied before in many dimensions. Because sports organizations have long been known for their ability to provide societal advantages such as enhanced physical health, education, and social integration, the concept of corporate social responsibility (CSR) has grown in popularity across the board (Jarvie, 2003; Myers et al., 2004; Lambourne, 2006). Sports organizations differ from one another and from other enterprises in terms of core operations, organizational structures, mindsets, and strategies, therefore CSR perception in sporting settings, particularly clubs, is vital for managers and sports management personnel (Kolyperas et al., 2015). The results showed that such perceived CSR can influence injury prevention expectations, injury risk perception and health up-gradation. The reason behind this association is supported by the fact that people become more aware of the hazards associated with sports activities when proper utilization of CSR is prevailing in their respective organizations. Previously very few studies tried to find the association between motivation for sports and these factors including injury prevention expectation, injury risk perception and health system and also found significant results (Perera et al., 2019). The research by Perera et al. (2019) provided motivation for assessing this kind of association in current research. This notion got strong support from their study. This research also tries to find the direct association of sports safety measures with injury prevention expectations, injury risk perceptions and health up-gradation of people involved in sports activities. The results were quite similar to the previous association between perceived CSR and these outcomes. It showed that proper safety measures provided by the sports organizations to their players and audiences lead to a better sense of injury prevention and the risks of sports-related injuries. It could also play a role in the upgradation of the health of the people involved. Previously, in this context, research was not carried out but supportive investigations also proved possible interactions like these (Ross et al., 2021). This research also evaluated the mediating role of sports safety measures taken out by sports organizations and communities involved in sports activities. The mediation between all three indirect relationships including perceived CSR, injury prevention expectations, injury risk perception and health up-gradation showed significant and positive results. This indicated that proper provision of sports safety measures by the organizations enhances the direct relationships of perceived CSR with injury prevention expectations, injury risk perception and health up-gradation. This proved its mediating role which helps in boosting these relationships. Previously, no study has found the mediating role of sports safety measures before for the sports activities, but such physical activities have been explored as a mediator between fitness-related factors (Camiletti-Moirón et al., 2020). Frontiers in Psychology | www.frontiersin.org The crisis of COVID-19 adversely affected the sports industry. Additionally, this health crisis influenced and amended the actions and management of the local sports bodies. However, researchers found that corporate social responsibility has a positive influence on their perceptions, expectations, and health. Therefore, the study intends to explore the impact of perceived corporate social responsibility and sports safety measures on injury prevention expectation, injury risk perception, and health up-gradation with the mediation of sports safety measures among sportsmen of local sports bodies. The outcome of the analysis showed that perceived corporate social responsibility leads to injury prevention expectation, injury risk perception, and health up-gradation. Also, the study found that sports safety measure mediates the relationship between perceived corporate social responsibility and injury prevention expectation, between perceived corporate social responsibility and injury risk perception, and between perceived corporate social responsibility and health up-gradation among sportsmen of local sports bodies. Theoretically, present examination expanded the research paradigm related to the impact of perceived corporate social responsibility on a major global crisis (COVID-19). In the sports literature, this investigation provided an in-depth understanding of PCSR on different factors such as injury prevention expectation, injury risk perception, and health up-gradation. Corporate social responsibility practices bring strategic advantages for sportsmen. In the recent era, the influence of CSR has received interest of the scholars and practitioners for motivating the sportsmen and inducing sportsmen spirit. Moreover, the mediating effect of sports safety measures has not been explored before, therefore this study would provide a deep insight into this concept and phenomenon for the readers. Most of the studies on CSR and its effect have been investigated in the context of multinational organizations and their employees; however, very few have examined CSR in the context of sportsmen, especially in the COVID-19 crisis. Thus, this theory thoroughly added value to the sports literature. Practically, local sports bodies should actively develop strategies to adopt and implement corporate social responsibility initiatives. Such programs lead to better sports performance of the sportsmen through injury prevention expectation, injury risk perception, and health up-gradation. The managers of the local sports bodies should engage in CSR initiatives such as environmental protection and donations. Not only this, the local bodies should encourage the sportsmen to participate in such programs and be socially responsible. In the context of global crises such as COVID-19, the local sports body managers should organize and promote socially responsible efforts such as charitable actions among the sportsmen. These actions help to support the areas that are hardest hit by the COVID-19 crisis. Additionally, the managers of local sports bodies should engage communities, local governments, and non-governmental organizations to develop response plans, strengthen prevention 11 June 2022 | Volume 13 | Article 919254 Ma et al. Corporate Social Responsibility and Safety of Sports DATA AVAILABILITY STATEMENT and safety measures, and monitor pandemic risk messages for better sports performance. The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. LIMITATIONS AND RECOMMENDATIONS The present study has a few limitations. First, the study was conducted using a small sample size; therefore, larger sample size can provide more generalized results. Results from a smaller sample size with convenience sampling can induce some biases in the results. These results can be verified with higher sample size in the European country where sports is religiously followed. This would help in comparing the results in Asia and Europe. Moreover, the present study was conducted in China, thus future studies can be conducted in different areas and regions. Another limitation of the study is the research design of the study. The study undertook a cross-sectional study design. Future studies can use a longitudinal study design in order to examine the effect of the variables in two different time periods. The study examined the role of perceived corporate social responsibility on injury prevention expectation, injury risk perception, and health up-gradation with the mediation of sports safety measures. Future studies incorporate the dimensions of CSR in the model and can examine the effect of CSR on the sports performance of sportsmen. ETHICS STATEMENT Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. 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Cartil. 23, 1122–1129. doi: 10.1016/j.joca.2015.02.021 Winship, C., and Zhuo, X. (2020). Interpreting t-statistics under publication bias: rough rules of thumb. J. Quant. Criminol. 36, 329–346. doi: 10.1007/s10940-018-9387-8 Woods, J. A., Hutchinson, N. T., Powers, S. K., Roberts, W. O., Gomez-Cabrera, M. C., Radak, Z., et al. (2020). The COVID-19 pandemic and physical activity. Sport. Med. Heal. Sci. 2, 55–64. doi: 10.1016/j.smhs.2020.05.006 Yingfei, Y., Mengze, Z., Zeyu, L., Ki-Hyung, B., Avotra, A. A. R. N., and Nawaz, A. (2021). Green logistics performance and infrastructure on service trade and Frontiers in Psychology | www.frontiersin.org 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. 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STUDIA MATHEMATICA 213 (3) (2012) Medians, continuity, and vanishing oscillation by Jonathan Poelhuis and Alberto Torchinsky (Bloomington, IN) Abstract. We consider properties of medians as they pertain to the continuity and vanishing oscillation of a function. Our approach is based on the observation that medians are related to local sharp maximal functions restricted to a cube of Rn . In considering the problem of the resistance of materials to certain types of deformations, F. John was led to the study of quasi-isometric mappings. The setting is essentially as follows. Let Q0 ⊂ Rn be a cube and f a continuous function on Q0 . Assume that to each subcube Q of Q0 with sides parallel to those of Q0 there is assigned a constant cQ and let µQ be the function of the real variable M given by |{y ∈ Q : |f (y) − cQ | > M }| µQ (M ) = . |Q| Let φ(M ) = supQ⊂Q0 µQ (M ), 0 < s < 1/2, and λ a number such that φ(λ) ≤ s. Then under these assumptions φ(M ) ≤ Ae−BM/λ holds for all nonnegative M where A, B are universal functions of s and the dimension n. Thus the space of functions of bounded mean oscillation (BMO) was introduced and the John–Nirenberg inequality established [5]. Strömberg [11] adopted this setting when studying spaces close to BMO and discussed different ways of describing the oscillation of a function f on cubes. He also incorporated the value s = 1/2 above, which corresponds to the notion of median value mf (Q) = mf (1/2, Q) of f over Q. The local maximal functions of Strömberg are of particular interest because they allow for pointwise estimates for Calderón–Zygmund singular integral operators [4, 6]. In this paper we consider properties of medians as they pertain to the continuity and vanishing oscillation of a function. Our approach is based on 2010 Mathematics Subject Classification: Primary 42B25; Secondary 46E30. Key words and phrases: medians, local sharp maximal function. DOI: 10.4064/sm213-3-3 [227] c Instytut Matematyczny PAN, 2012 228 J. Poelhuis and A. Torchinsky the observation that medians are related to local sharp maximal functions restricted to a cube Q0 ⊂ Rn with parameter 0 < s ≤ 1/2 by means of the expression ],φ f (x) = M0,s,Q 0 ∼ sup inf x∈Q, Q⊂Q0 c sup x∈Q, Q⊂Q0 m|f −c| (1 − s, Q) φ(|Q|) m|f −mf (1−s,Q)| (1 − s, Q) φ(|Q|) , where φ equals 1 in the case of functions with bounded median oscillation with parameter s (bmos ), and satisfies appropriate conditions in the case of functions with vanishing median oscillation with parameter s (vmos ). Strömberg showed that bmos = BMO, and, similarly, we show here that for sufficiently small s, vmos = VMO, the space of functions of vanishing mean oscillation. Moreover, since VMO is known to contain bounded discontinuous functions [8, 10], we complete the picture by giving criteria for continuity of functions equivalent to a bounded function on a cube in terms of medians. The paper is organized as follows. In Section 1 we introduce the notions of median and maximal median with respect to a parameter 0 < s < 1; when s 6= 1/2 we refer to these medians as biased with parameter s. In Section 2 we consider the a.e. convergence of maximal biased medians in the spirit of Fujii’s results [3] for s = 1/2. In Section 3, motivated by similar results involving averages [9], we characterize continuity in terms of maximal biased medians with parameter > 1/2. In Section 4 we extend the Strömberg decomposition of cubes to parameters > 1/2. Finally, in Section 5 we consider the spaces of functions with vanishing median oscillation, establish a John–Nirenberg type inequality they satisfy, and show that, as anticipated, they coincide with VMO for sufficiently small s. 1. Medians and maximal medians. In what follows we restrict our attention to cubes with sides parallel to the coordinate axes. Definition 1.1. For a cube Q ⊂ Rn , 0 < s < 1, and a real-valued measurable function f on Q we say that mf (s, Q) is a median value of f over Q with parameter s if (1.1) |{y ∈ Q : f (y) < mf (s, Q)}| ≤ s|Q| and (1.2) |{y ∈ Q : f (y) > mf (s, Q)}| ≤ (1 − s)|Q|. When s = 1/2, mf (1/2, Q) = mf (Q) corresponds to a median value of f over Q. The set of median values of f is one point or a closed interval as the example f = χ[1/2,1) on [0, 1] shows. It is therefore convenient to work Medians, continuity, and vanishing oscillation 229 with maximal medians, which are uniquely defined [1, 3]. More precisely, we have Definition 1.2. For a cube Q ⊂ Rn , 0 < s < 1, and a real-valued measurable function f on Q, we say that Mf (s, Q) is the maximal median of f over Q with parameter s if Mf (s, Q) = sup{M : |{y ∈ Q : f (y) < M }|} ≤ s|Q|. The reader will have no difficulty in proving the sup above is assumed, that is to say, |{y ∈ Q : f (y) < Mf (s, Q)}| ≤ s|Q|. To justify the nomenclature of maximal median we verify that Mf (s, Q) satisfies the conditions that characterize medians. (1.1) is guaranteed since Mf (s, Q) is the maximum value for which it holds. As for (1.2), let Bn = {y ∈ Q : f (y) ≥ Mf (s, Q) + 1/n} and note that |Bn | ≤ (1 − s)|Q|, all n, and {y ∈ Q : f (y) > Mf (s, Q)} ⊂ lim inf n Bn . Then |{y ∈ Q : f (y) > Mf (s, Q)}| ≤ lim inf n |Bn | ≤ (1 − s)|Q|. Hereafter when considering a median we mean the maximal median and denote it simply by mf (s, Q). Clearly maximal medians satisfy |{y ∈ Q : f (y) ≤ mf (s, Q)}| ≥ s|Q| (1.3) and (1.4) |{y ∈ Q : f (y) ≥ mf (s, Q)}| ≥ (1 − s)|Q|. We summarize the basic properties of maximal medians that are of interest to us in the following proposition. Proposition 1.1. Let Q ⊂ Rn be a cube, 0 < s, t < 1, and f, g realvalued measurable functions on Q. Then the following properties hold: (i) For s < t, mf (s, Q) ≤ mf (t, Q). (1.5) (ii) If f ≤ g a.e., then mf (s, Q) ≤ mg (s, Q). (1.6) (iii) For a constant c, (1.7) mf (s, Q) − c = mf −c (s, Q). (iv) If f, g ≥ 0 a.e., 0 < s, s1 < 1, and 0 < t < s + s1 − 1, then (1.8) mf +g (t, Q) ≤ mf (s, Q) + mg (s1 , Q). (v) In general, m−|f | (s, Q) ≤ mf (s, Q) ≤ m|f | (s, Q). And if mf (s, Q) ≤ 0, then (1.9) |mf (s, Q)| ≤ m|f | (1 − s, Q). 230 J. Poelhuis and A. Torchinsky Thus for general f , (1.10) |mf (s, Q)| ≤ m|f | (s, Q), 1/2 ≤ s < 1. (vi) If f ≥ 0 is locally integrable and fQ denotes the average of f over Q, then 1 (1.11) mf (s, Q) ≤ fQ . 1−s Proof. (i) Since |{y ∈ Q : f (y) < mf (s, Q)}| ≤ s|Q| < t|Q|, (1.5) holds. (ii) Up to a set of measure zero {y ∈ Q : g(y) < mf (s, Q)} ⊂ {y ∈ Q : f (y) < mf (s, Q)}. Therefore |{y ∈ Q : g(y) < mf (s, Q)}| ≤ s|Q|, and so mf (s, Q) ≤ mg (s, Q). (iii) Since {y ∈ Q : f (y) < mf (s, Q)} = {y ∈ Q : f (y)−c < mf (s, Q)−c} it readily follows that mf (s, Q) − c ≤ mf −c (s, Q). And since {y ∈ Q : f (y)−c < mf −c (s, Q)} = {y ∈ Q : f (y) < mf −c (s, Q)+c}, mf −c (s, Q)+c ≤ mf (s, Q). Note that, in particular, mc (s, Q) = c. (iv) For the sake of argument suppose that f, g are measurable functions on Q such that mf +g (t, Q) − (mf (s, Q) + mg (s1 , Q)) > 2η > 0. Then {y ∈ Q : f (y) < mf (s, Q)+η}∩{y ∈ Q : g(y) < mg (s1 , Q)+η} ⊂ {y ∈ Q : f (y)+ g(y) < mf +g (t, Q)}. Now, since |{y ∈ Q : f (y) < mf (s, Q) + η}| ≥ s|Q|, |{y ∈ Q : g(y) < mg (s1 , Q) + η}| ≥ s1 |Q|, and |{y ∈ Q : f (y) + g(y) < mf (t, Q)}| ≤ t|Q|, it readily follows that s + s1 ≤ 1 + t, which is not the case. (v) Since −|f | ≤ f ≤ |f |, by (1.6), m−|f | (s, Q) ≤ mf (s, Q) ≤ m|f | (s, Q). Now, if mf (s, Q) ≤ 0 note that {y ∈ Q : |f (y)| < −mf (s, Q)} = {y ∈ Q : mf (s, Q) < −|f (y)|} ⊂ {y ∈ Q : mf (s, Q) < f (y)}, and therefore |{y ∈ Q : |f (y)| < −mf (s, Q)}| ≤ (1−s)|Q|. Consequently, |mf (s, Q)| ≤ m|f | (1−s, Q). And since for s ≥ 1/2, 1 − s ≤ s, by (1.5) we have |mf (s, Q)| ≤ m|f | (s, Q) for that range of s. (vi) We may assume that mf (s, Q) 6= 0. Then by (1.4) and Chebyshev’s inequality,  1 f (y) dy, (1 − s)|Q| ≤ |{y ∈ Q : f (y) ≥ mf (s, Q)}| ≤ mf (s, Q) Q and the conclusion follows. The restriction 1/2 ≤ s < 1 is necessary for (1.10) to hold. Let Q = [0, 1] and f (x) = −2χ[0,1/2) (x) + χ[1/2,1) (x); then for 0 < s < 1/2, mf (s, Q) = −2 but m|f | (s, Q) = 1 < 2. And, in contrast to averages, the restriction 0 < t < s + s1 − 1 is necessary for (1.8) to hold. To see this let Q = [0, 1], and pick 1/2 < s1 ≤ s < 1, and t = s + s1 − 1 > 0. If f = χ[0,1−s] and g = χ[1−s1 ,2(1−s1 )] , {y ∈ Q : f (y) + g(y) < 1} = (1 − s, 1 − s1 ) ∪ (2(1 − s1 ), 1] has measure (s − s1 ) + 1 − 2(1 − s1 ) = t, and therefore, although mf (s, Q) = mg (s1 , Q) = 0, mf +g (t, Q) = 1. Medians, continuity, and vanishing oscillation 231 Finally, maximal medians can be expressed in terms of distribution functions or nonincreasing rearrangements. Recall that the nonincreasing rearrangement f ∗ of f at level λ > 0 is given by f ∗ (λ) = inf{α > 0 : |{x ∈ Rn : |f (x)| > α}| ≤ λ} and satisfies (1.12) |{y ∈ Rn : |f (y)| > f ∗ (u)}| ≤ u, u > 0. We then have Proposition 1.2. Let Q ⊂ Rn be a cube, 0 < s < 1, and f a measurable function on Q. Then m|f | (1 − s, Q) = inf{α > 0 : |{y ∈ Q : |f (y)| > α}| < s|Q|} = (f χQ )∗ (s|Q|). Proof. Let α = inf{α > 0 : |{y ∈ Q : |f (y)| > α}| < s|Q|}. Then for all ε > 0 it readily follows that |{y ∈ Q : |f (y)| ≤ α + ε}| > (1 − s)|Q|, which together with (1.3) implies m|f | (1 − s, Q) ≤ α + ε. Thus m|f | (1 − s, Q) ≤ α. Next, by (1.12), |{y ∈ Q : |f (y)| > (f χQ )∗ (s|Q|)}| ≤ s|Q|, which gives α ≤ (f χQ )∗ (s|Q|). Finally, since for ε > 0, |{y ∈ Q : |f (y)| > (f χQ )∗ (s|Q|) − ε}| > s|Q|, by (1.2) it readily follows that (f χQ )∗ (s|Q|) − ε < m|f | (1 − s, Q), and consequently (f χQ )∗ (s|Q|) ≤ m|f | (1 − s, Q). Other equivalent expressions appearing in the literature include those in [2]. 2. Convergence of medians. The examples following Proposition 1.1 suggest that medians rely more heavily on the distribution of the values of f than do averages. On the other hand, averages and medians are not always at odds. In particular, using (1.11) the reader should have no difficulty in verifying that the following version of the Lebesgue differentiation theorem holds: If f is a locally integrable function on Rn and 1/2 ≤ s < 1, then lim x∈Q, Q→x mf (s, Q) = f (x) at every Lebesgue point x of f . Thus, in some sense mf (s, Q) is a good substitute for fQ for small Q. In fact, a more careful argument gives that the biased maximal medians mf (s, Q) of an arbitrary measurable function f converge to f a.e., a fact observed by Fujii [3] for the case s = 1/2. Theorem 2.1. Let f be a real-valued, finite a.e. measurable function on Rn , and 0 < s < 1. Then (2.1) lim x∈Q, Q→x mf (s, Q) = f (x) a.e. In particular, (2.1) holds at every point of continuity x of f . 232 J. Poelhuis and A. Torchinsky n : (j − 1)/2k ≤ Proof. For k ≥ 1 and an integer j, let Ek,j = {x ∈ PR ∞ k k f (x) < j/2 }, ak,j = (j − 1)/2 , and S put Sk (x) = j=−∞ ak,j χEk,j (x). Note that since f is finite a.e., Rn = k,j Ek,j except possibly for a set of measure 0, and when f (x) is finite we have 0 ≤ f (x) − Sk (x) ≤ 2−k , all cubes Q. Let which gives mSk (s, Q) ≤ mf (s, Q) ≤ mSk (s, Q) + 2−k for S∞ Ak,j = {x ∈ Ek,j : x is a point of density for Ek,j }, Ak = j=−∞ Ak,j . Since S n f is finite a.e., |Rn \ Ak | = 0 for all k, and if A = ∞ k=1 Ak , also |R \ A| = 0. We claim that the limit in question exists for x ∈ A. Given ε > 0, pick k such that 2−k+1 < ε. Then x ∈ Ak,j for some j, and |Ak,j ∩ Q| = 1. x∈Q, Q→x |Q| lim Let δ = max{s, 1 − s} and note that for all cubes Q with small enough measure containing x, |Ak,j ∩ Q| > δ. |Q| We restrict our attention to such small cubes Q containing x. Note that for these cubes mSk (s, Q) = ak,j . Indeed, on the one hand, since Sk (y) = ak,j for y ∈ Ak,j , |{y ∈ Q : Sk (y) < ak,j }| ≤ |Ack,j ∩ Q| < s|Q|, and therefore ak,j ≤ mSk (s, Q). And, on the other, since for ε > 0, {y ∈ Q : Sk (y) < ak,j+ε} ⊃ Ak,j ∩ Q, it follows that |{y ∈ Q : Sk (y) < ak,j + ε}| ≥ |Ak,j ∩ Q| ≥ s|Q|. Hence, mSk (s, Q) ≤ ak,j + ε, and since ε is arbitrary, mSk (s, Q) ≤ ak,j . Then, since ak,j = mSk (s, Q) = Sk (x) for x ∈ Ak,j , |mf (s, Q) − f (x)| ≤ |mf (s, Q) − mSk (s, Q)| + |mSk (s, Q) − f (x)| ≤ 2−k + (f (x) − Sk (x)) ≤ 2−k+1 < ε. In other words, |mf (s, Q) − f (x)| < ε for x ∈ A and all Q with small enough measure containing x. Now, at a point of continuity x of f , given ε > 0, let δ > 0 be such that |f (y) − f (x)| ≤ ε for y ∈ B(x, δ). Then for y in a cube Q containing x and contained in B(x, δ) we have −ε ≤ f (y) − f (x) ≤ ε, and consequently −ε = m−ε (s, Q) ≤ mf −f (x) (s, Q) = mf (s, Q) − f (x) ≤ mε (s, Q) = ε, hence |mf (s, Q) − f (x)| ≤ ε. 3. A median characterization for continuity. We say that a measurable function f on a cube Q0 ⊂ Rn is equivalent to a continuous function on Q0 if the values of f can be modified on a set of Lebesgue measure 0 so as to coincide with a continuous function on Q0 ; similarly for f equivalent to a bounded function on a cube. In this section we characterize those measurable functions equivalent to a bounded function on a cube that are equivalent to a continuous function on that cube in terms of medians, keeping in mind Medians, continuity, and vanishing oscillation 233 that in the case of locally integrable functions the condition involves the consideration of oscillations involving two nonoverlapping cubes [9]. Definition 3.1. For 0 < s < 1 and nonoverlapping cubes Q1 , Q2 ⊂ Rn , let Ψs (f, Q1 , Q2 ) = |Q1 | |Q2 | mf (s, Q1 ) + mf (s, Q2 ), |Q1 ∪ Q2 | |Q1 ∪ Q2 | and Ω(f, s, δ) = sup inf Ψs (|f − c|, Q1 , Q2 ). diam(Q1 ∪Q2 )≤δ c Ψs is a weighted average of maximal medians of f in the spirit of averages and Ω(f, s, δ) is related to the oscillation of a measurable function on a cube, as shown by the following result. Theorem 3.1. Let Q0 ⊂ Rn be a cube, 1/2 < s < 1, f a measurable function on Q0 that is equivalent to a bounded function there, and ω(f, δ), δ > 0, the essential modulus of continuity of f defined by   ω(f, δ) = sup ess sup |f (x + h) − f (x)| . |h|≤δ x,x+h∈Q0 Then we have Ω(f, s, δ) = ω(f, δ)/2. Proof. For nonoverlapping cubes Q1 , Q2 ⊂ Q0 , let Θ = ess osc(f, Q1 ∪ Q2 ) = ess sup f − ess inf Q1 ∪Q2 f. Q1 ∪Q2 Let δ > 0. If x ∈ Q1 ∪ Q2 ⊂ Q0 is such that x + h ∈ Q0 where |h| ≤ δ, since ess sup |f (x + h) − f (x)| ≥ ess sup f − ess inf f, Q1 ∪Q2 x,x+h∈Q0 Q1 ∪Q2 taking the sup over |h| ≤ δ it readily follows that ω(f, δ) ≥ Θ. Moreover, since for y ∈ Q1 and an arbitrary constant c, n o |f (y) − c| ≤ max ess sup f − c, c − ess inf f , Q1 ∪Q2 Q1 ∪Q2 picking c = (sup inf Q1 ∪Q2 f + ess inf Q1 ∪Q2 f )/2, it follows that |f (y) − c| ≤ Θ/2, and, consequently, m|f −c| (s, Q1 ) ≤ mΘ/2 (s, Q1 ) = Θ/2; similarly we have m|f −c| (s, Q2 ) ≤ Θ/2. Therefore, inf Ψs (|f − c|, Q1 , Q2 ) ≤ c Θ |Q2 | Θ Θ |Q1 | + = , |Q1 ∪ Q2 | 2 |Q1 ∪ Q2 | 2 2 and consequently Ω(f, s, δ) ≤ Θ/2 ≤ ω(f, δ)/2. Conversely, let 0 < t < 2s − 1. Then for fixed δ > 0, given ε > 0, pick h with |h| < δ such that ess supx,x+h∈Q0 |f (x + h) − f (x)| ≥ ω(f, δ) − ε. Then E = {x ∈ Q0 : x + h ∈ Q0 and |f (x + h) − f (x)| ≥ ω(f, δ) − ε} has positive 234 J. Poelhuis and A. Torchinsky measure. Let x ∈ E be a point of density of E and a small enough so that Q(x, a), Q(x + h, a) are nonoverlapping and |E ∩ Q(x, a)| > 1 − t. |Q(x, a)| Now, since {y ∈ Q(x + h, a) : g(y) < M } = {y ∈ Q(x, a) : g(y + h) < M } and |Q(x, a)| = |Q(x + h, a)|, it readily follows that m|f −c| (s, Q(x + h, a)) = m|f (· +h)−c| (s, Q(x, a)), and consequently, since |f (y+h)−f (y)| ≤ |f (y+h)−c| + |f (y) − c|, by (1.8) and (1.6), m|f −c| (s, Q(x, a)) + m|f −c| (s, Q(x + h, a)) ≥ m|f −c|+|f (· +h)−c| (t, Q(x, a)) ≥ m|f (· +h)−f | (t, Q(x, a)). Therefore, Ψs (|f − c|, Q(x, a), Q(x + h, a)) 1 1 ≥ m|f −c| (s, Q(x, a)) + m|f −c| (s, Q(x + h, a)) 2 2 1 ≥ m|f (· +h)−f | (t, Q(x, a)). 2 Finally, since |E ∩ Q(x, a)| = |{y ∈ Q(x, a) : |f (y + h) − f (y)| ≥ ω(f, δ) − ε}| > (1 − t)|Q(x, a)|, it readily follows that m|f (· +h)−f | (t, Q(x, a)) ≥ ω(f, δ) − ε, which, since ε is arbitrary, √ implies Ψs (|f − c|, Q(x, a), Q(x + h, a)) ≥ ω(f, δ)/2. Thus Ω(f, s, δ + ( 2a)n ) ≥ ω(f, δ), and letting a → 0, Ω(f, s, δ) ≥ ω(f, δ)/2. Theorem 3.2. Let Q0 ⊂ Rn be a cube, 1/2 < s < 1, and f a measurable function on Q0 that is equivalent to a bounded function there. Then f is equivalent to a continuous function on Q0 iff limη→0+ Ω(f, s, η) = 0. The proof follows at once from Theorem 3.1. Note that by Proposition 1.2 the conclusion can also be stated in terms of rearrangements. 4. A decomposition of cubes. Strömberg’s essential tool in dealing with the oscillation of functions and local maximal functions is a decomposition of cubes [11]. In this section we extend the results to biased medians with parameters > 1/2. We begin by introducing the local sharp maximal function restricted to a cube. Definition 4.1. Let Q0 ⊂ Rn and 0 < s ≤ 1/2. For a measurable function f on Q0 , the local sharp maximal function restricted to Q0 of f , ] written M0,s,Q f (x), is defined at x ∈ Q0 as 0 (4.1) ] M0,s,Q f (x) = 0 sup inf inf{α ≥ 0 : |{y ∈ Q : |f (y) − c| > α}| < s|Q|}. x∈Q, Q⊂Q0 c ] ] When Q0 = Rn , M0,s,R n f (x) = M0,s f (x) denotes the local sharp maximal n function of f at x ∈ R . Medians, continuity, and vanishing oscillation 235 ] The range 0 < s ≤ 1/2 is necessary since for s > 1/2, M0,s,Q f (x) = 0 for a function f that takes two different values. Local maximal functions, as well as maximal functions defined in terms of rearrangements, can be expressed in terms of medians. Let ωs (f, Q) = inf c ((f − c)χQ )∗ (s|Q|). Then by Proposition 1.2, ] f (x) = M0,s,Q 0 sup ωs (f, Q) = x∈Q, Q⊂Q0 sup inf m|f −c| (1 − s, Q). x∈Q, Q⊂Q0 c The first expression above is used by Lerner [6, 7]. An efficient choice for c in the infimum above is m|f −mf (1−s,Q)| (1 − s, Q). Indeed, for Q ⊂ Q0 and a constant c, since 1 − s ≥ 1/2, by (1.7) and (1.10), |mf (1 − s, Q) − c| ≤ m|f −c| (1 − s, Q). (4.2) Then, since |f (y) − mf (1 − s, Q)| ≤ |f (y) − c| + |c − mf (1 − s, Q)|, by (1.5), (1.7), and (4.2), m|f −mf (1−s,Q)| (1 − s, Q) ≤ m|f −c| (1 − s, Q) + |c − mf (1 − s, Q)| ≤ m|f −c| (1 − s, Q) + m|f −c| (1 − s, Q) = 2m|f −c| (1 − s, Q), and consequently inf m|f −c| (1 − s, Q) ≤ m|f −mf (1−s,Q)| (1 − s, Q) (4.3) c ≤ 2 inf m|f −c| (1 − s, Q). c The decomposition of cubes relies on three lemmas which we prove next. Lemma 4.1. Let Q ⊂ Rn be a cube, 0 < s ≤ 1/2, 1/2 ≤ t ≤ 1 − s, and f a measurable function on Q. Then for any η > 0, (4.4) ] |{y ∈ Q : |f (y) − mf (t, Q)| ≥ 2 inf M0,s,Q f (x) + η}| < s|Q|. x∈Q Proof. For fixed c, let α(c) = m|f −c| (1 − s, Q). Then by (4.2) and (1.5), (4.5) |mf (t, Q) − c| ≤ m|f −c| (t, Q) ≤ m|f −c| (1 − s, Q) = α(c), and by (1.4), (4.6) |{y ∈ Q : |f (y) − c| ≥ α(c) + ε}| < s|Q|, ε > 0. Let m = inf c α(c) and pick {ck } such that m ≤ α(ck ) ≤ m + 1/k, all k. Then by (4.5), |f (y) − ck | ≥ |f (y) − mf (t, Q)| − |mf (t, Q) − ck | ≥ |f (y) − mf (t, Q)| − α(ck ), and consequently, since 2m + η ≥ α(ck ) + (η − 2/k), {y ∈ Q : |f (y) − mf (t, Q)| ≥ 2m + η} ⊂ {y ∈ Q : |f (y) − ck | ≥ α(ck ) + εk }, where we have chosen k sufficiently large so that εk = η − 2/k > 0. Then by (4.6), 236 J. Poelhuis and A. Torchinsky # |{y ∈ Q : |f (y)−mf (t, Q)| > 2m+η}| < s|Q|. Finally, since M0,s,Q f (x) ≥ m for all x ∈ Q, (4.4) holds. Lemma 4.2. Let Q ⊂ Rn be a cube, 0 < s ≤ 1/2, 1/2 ≤ t ≤ 1 − s, η > 0, and f a measurable function on an open cube containing Q. Then for any family of cubes {Qε } with (1 − ε)Q ⊂ Qε ⊂ (1 + ε)Q, ] lim sup |mf (t, Q) − mf (t, Qε )| ≤ 2 inf M0,s,Q f (x) + η. x∈Q ε→0+ ] f (x). For the sake of argument assume Proof. Let A = inf x∈Q M0,s,Q there is a sequence εk → 0 such that |mf (t, Q) − mf (t, Qεk )| > 2A + η for all k. Then by (1.10), 2A + η < |mf (t, Q) − mf (t, Qεk )| ≤ m|f −mf (t,Q)| (t, Qεk ), and consequently, by (1.4), (4.7) |{y ∈ Qεk : |f (y) − mf (t, Q)| > 2A + η}| ≥ (1 − t)|Qεk |. Since Qεk ⊂ (1 + εk )Q, the left-hand side of (4.7) is bounded above by |{y ∈ (1 + εk )Q : |f (y) − mf (t, Q)| > 2A + η}| ≤ ((1 + εk )n − 1)|Q| + |{y ∈ Q : |f (y) − mf (t, Q)| > 2A + η}|, and since (1 − εk )Q ⊂ Qεk , the right-hand side of (4.7) is bounded below by (1 − t)(1 − εk )n |Q|. Combining these estimates it follows that |{y ∈ Q : |f (y)−mf (t, Q)| > 2A+η}| ≥ ((1−t)(1−εk )n −((1+εk )n −1))|Q|. Now, by (4.4) there exists δ > 0 such that |{y ∈ Q : |f (y) − mf (t, Q)| ≥ 2A + η}| = (s − δ)|Q|, and so s − δ ≥ ((1 − t)(1 − εk )n − ((1 + εk )n − 1)). Thus, letting k → ∞ implies s − δ ≥ 1 − t ≥ s, which is not the case. Lemma 4.3. Let Q0 , Q1 ⊂ Rn be cubes with Q0 ⊂ Q1 and |Q1 | ≤ 2k |Q0 | for some integer k, 0 < s ≤ 1/2, 1/2 ≤ t ≤ 1 − s, and f a measurable function on Q1 . Then (4.8) ] |mf (t, Q0 ) − mf (t, Q1 )| ≤ 10k inf M0,s,Q f (x). 1 x∈Q0 Proof. By the triangle inequality it suffices to prove the case k = 1. Let ] A = inf x∈Q0 M0,s,Q f (x). For the sake of argument suppose that (4.8) does 1 not hold. Then if A > 0, by Lemma 4.2, for any fixed 0 < η < A/2, there exists a cube Q2 such that Q0 ⊂ Q2 ⊂ Q1 and |mf (t, Q2 ) − mf (t, Q0 )| > 4A + 2η, |mf (t, Q2 ) − mf (t, Q1 )| > 4A + 2η. Medians, continuity, and vanishing oscillation 237 And if A = 0, then |mf (t, Q0 ) − mf (t, Q1 )| > 0 and there exists a cube Q2 such that Q0 ⊂ Q2 ⊂ Q1 and |mf (t, Q2 ) − mf (t, Q0 )| > 2η, |mf (t, Q2 ) − mf (t, Q1 )| > 2η for η sufficiently small. Thus in both cases the sets {y ∈ Qk : |f (y) − mf (t, Qk )| ≤ 2A + η}, k = 0, 1, 2, are pairwise disjoint subsets of Q1 , and consequently {y ∈ Q0 : |f (y) − mf (t, Q0 )| ≤ 2A + η} ∪ {y ∈ Q2 : |f (y) − mf (t, Q2 )| ≤ 2A + η} ⊂ {y ∈ Q1 : |f (y) − mf (t, Q1 )| > 2A + η}. ] Therefore, since inf x∈Qk M0,s,Q f (x) ≤ A for k = 0, 1, 2, by Lemma 4.1, k (1 − s)|Q0 | + (1 − s)|Q2 | < s|Q1 |. Thus 2(1 − s)|Q0 | < s|Q1 | < 2s|Q0 |, and consequently 1 < 2s, which is not the case. We are now ready to consider the decomposition of cubes relative to medians. Proposition 4.1. Let Q ⊂ Rn be a cube, 0 < s ≤ 1/2, 1/2 ≤ t ≤ 1 − s, δ, β > 0, and f a measurable function on Q. Then if |mf (t, Q)| ≤ δ, there exists a (possibly empty) family {Qk } of nonoverlapping dyadic subcubes of Q so that ] f (y) > β}, (1) Qk 6⊂ {y ∈ Q : M0,s,Q (2) δ < |mf (t, Qk )| ≤ δ + 10nβ, S # f (y) > β} ∪ k Qk ). (3) |f (x)| ≤ δ for a.e. x ∈ Q \ ({y ∈ Q : M0,s,Q ] Proof. If M0,s,Q f (y) > β for all y ∈ Q we pick {Qk } as the empty family. Otherwise subdivide Q dyadically into 2n subcubes and note that by Lemma ] 4.3, for each dyadic subcube Q0 , |mf (t, Q0 )| ≤ δ + 10n inf x∈Q0 M0,s,Q f (x). 0 Thus for each of these subcubes Q one of the following holds: ] (a) Q0 ⊂ {y ∈ Q : M0,s,Q f (y) > β}: we discard Q0 . 0 (b) Q satisfies conditions (1) and (2) above: we collect this Q0 . ] (c) Q0 6⊂ {y ∈ Q : M0,s,Q f (y) > β} but |mf (t, Q0 )| ≤ δ: we subdivide Q0 and continue in this fashion. S ] Finally, a.e. x ∈ Q \ ({y ∈ Q : M0,s,Q f (y) > β} ∪ k Qk ) is contained in arbitrarily small cubes {Qk (x)} containing x so that |mf (t, Qk (x))| ≤ δ. By Theorem 2.1 it readily follows that |f (x)| ≤ δ for a.e. such x. We can be more precise in the description of the cubes above when ] M0,s,Q f ∈ L∞ (Q). Observe that f − mf (t, Q) satisfies mf −mf (t,Q) (t, Q) = 0 ] ] and M0,s,Q f (x) = M0,s,Q (f − mt (t, Q))(x) for all x ∈ Q, which means that 238 J. Poelhuis and A. Torchinsky the decomposition for f − mf (t, Q) holds for any δ > 0. Let {Qj } and {Qk } denote the families of cubes obtained from the decomposition with ] parameters β ≥ kM0,s,Q f kL∞ (Q) and δ1 = 4β + 2η and δ2 = 2δ1 + 10nβ, respectively. S S Observe that by construction we have k Qk ⊂ j Qj . To see this consider a dyadic subcube Q0 of Q that has not been discarded; this depends on β and not on δ1 or δ2 . If Q0 is a Qj , then δ1 < |mf −mf (t,Q) (t, Q0 )| ≤ δ1 + 10nβ < δ2 and Q0 is not a Qk . So any Qk contained in Q0 arises from subsequent subdivisions of Q0 . On the other hand, if Q0 is not a Qj , then |mf −mf (t,Q) (t, Q0 )| ≤ δ1 < δ2 and Q0 is not a Qk either. Since this relation is maintained at every level of the successive dyadic subdivisions, the Qk ’s arise from subdivisions of Qj ’s. From here on the argument proceeds as in Lemma 4.3. Note that δ1 < |mf −mf (t,Q) (t, Qj )| = |mf (t, Qj ) − mf (t, Q)| ≤ δ1 + 10nβ = δ2 − δ1 , and δ2 < |mf −mf (t,Q) (t, Qk )| = |mf (t, Q) − mf (t, Qk )|. Therefore, |mf (t, Qj ) − mf (t, Qk )| ≥ |mf (t, Qk ) − mf (t, Q)| − |mf (t, Qj ) − mf (t, Q)| > δ2 − (δ2 − δ1 ) = δ1 = 4β + 2η. Thus it readily follows that the sets {y ∈ Q : |f (y)−mf (t, Q)| ≤ 2β +η}, {y ∈ Qj : |f (y) − mf (t, Qj )| ≤ 2β + η}, and {y ∈ Qk : |f (y) − mf (t, Qk )| ≤ 2β + η} are nonoverlapping, and so (4.9) {y ∈ Qj : |f (y) − mf (t, Qj )| ≤ 2β + η} ∪ {y ∈ Qk : |f (y) − mf (t, Qk )| ≤ 2β + η} ⊂ {y ∈ Q : |f (y) − mf (t, Q)| > 2β + η}. ] Now, since inf x∈Qj M0,s,Q f (x) ≤ β for each Qj , by (4.4) it follows that j X X (1 − s) |Qj | ≤ |{y ∈ Qj : |f (y) − mf (t, Qj )| ≤ 2β + η}|, j j and a similar estimate holds with the Qk ’s in place of the Qj ’s. Finally, since the sets on the left-hand side of (4.9) are pairwise disjoint for all j and k, S S ] and since k Qk ⊂ j Qj and inf x∈Q M0,s,Q f (x) ≤ β, by Lemma 4.1, X X X s 2 |Qk | ≤ |Qk | + |Qj | ≤ |Q|, 1−s k k j and consequently, since 2(1 − s) ≥ 1, X s (4.10) |Qk | ≤ |Q| ≤ s|Q|. 2(1 − s) k Medians, continuity, and vanishing oscillation 239 5. Vanishing median oscillation. We say that a measurable function f defined on a cube Q0 ⊂ Rn is of vanishing median oscillation with parameter s in Q0 (vmos (Q0 )) if φs (u) = inf m|f −c| (1 − s, Q) sup Q⊂Q0 , |Q|≤u c satisfies limu→0+ φs (u) = 0. Note that by (1.11), inf m|f −c| (1 − s, Q) ≤ c 1 1  |f (y) − fQ | dy, s |Q| Q and therefore limu→0+ φs (u) = 0 for all s whenever f ∈ VMO(Q0 ). Here we show that the spaces actually coincide for s ≤ 2−n . Now, φs is a nonnegative, nondecreasing continuous function that vanishes at the origin, and vmos may be described in terms of such functions φ as follows. Let m|f −mf (1−s,Q)| (1 − s, Q) m|f −c| (1 − s, Q) kf ks,φ,Q0 = sup inf ∼ sup , φ(|Q|) φ(|Q|) Q⊂Q0 c Q⊂Q0 and bmos,φ (Q0 ) = {f : f S is defined and measurable on Q0 , and kf ks,φ,Q0 < ∞}. Then vmos (Q0 ) = φ bmos,φ (Q0 ). Now fix Q0 and 0 < s ≤ 1/2n . Let φ : R+ → R+ be continuous, nondecreasing, and φ(0) = 0, and define Ψ|Q0 | : [0, 2n |Q0 |] → R+ by 2n |Q  0| (5.1) Ψ|Q0 | (u) = u φ(v) dv. v We can then prove a strengthened version of the John–Nirenberg inequality. Theorem 5.1. Let f ∈ bmos,φ (Q0 ) for some φ as above and Ψ|Q0 | (u) be given by (5.1). Then there exist constants c1 , c2 independent of f and of all subcubes Q ⊂ Q0 so that (5.2) −1 |{y ∈ Q : |f (y) − mf (1 − s, Q)| > λ}| ≤ c1 Ψ|Q| (c2 λ/kf ks,φ,Q ), λ > 0. ] Proof. If kf ks,φ,Q0 = 0, clearly M0,s,Q f (x) = 0 for all x ∈ Q0 and by 0 Lemma 4.3, the medians of f over all subcubes of Q0 are constant. Then by Theorem 2.1, f is a.e. constant, and the conclusion holds in this case. Otherwise, since kf − cks,φ,Q0 = kf ks,φ,Q0 and kcf ks.φ,Q0 = |c| kf ks,φ,Q0 for all constants c, we may assume that kf ks,φ,Q0 = 1 and mf (1 − s, Q0 ) = 0. Then by (4.3), m|f −mf (1−s,Q)| (t, Q) ≤ 2φ(|Q|) ≤ 2φ(|Q0 |) for all Q ⊂ Q0 , ] and consequently kM0,s,Q f kL∞ (Q0 ) ≤ 2φ(|Q0 |). Pick now β0 = 2φ(|Q0 |), 0 240 J. Poelhuis and A. Torchinsky and note that since φ(u) > 0 for u > 0, δ0 = (10n+9)β0 works in Proposition ] f (y) > β0 }| 4.1 and in the comments that follow it. Since |{y ∈ Q0 : M0,s,Q 0 1 = 0 we get a (first-generation) family {Qj } of nonoverlapping subcubes of Q0 so that (1) δ0 < |mf (t, Q1j )| ≤ δ0 + 10nβ0 for all j, S (2) |f (x)| ≤ δ0 for a.e. x ∈ Q0 \ j Q1j , and P 1 (3) j |Qj | ≤ s|Q0 |. Now we fix one cube Q1j of this family, which for simplicity we denote Q1 , and define g = f − mf (t, Q1 ). Note that mg (t, Q1 ) = 0 and g − mg (t, Q) = f − mf (t, Q) for all Q ⊂ Q1 . Then as above m|g−mg (t,Q)| (t, Q) ≤ 2φ(|Q|) for ] n all Q ⊂ Q1 , and thus kM0,s,Q 1 gkL∞ (Q1 ) ≤ 2φ(|Q|) = 2φ(|Q0 |/2 ). We then pick (first-generation) parameters β1 = 2φ(|Q0 |/2n ) and δ1 = ] (10n + 9)β1 , which gives |{y ∈ Q1 : M0,s,Q 1 g(y) > β1 }| = 0. As before, we get a (second-generation) nonoverlapping family {Q2j } ⊂ Q1 so that (1) δ1 < |mg (t, Q2j )| ≤ δ1 + 10nβ1 for all j, S (2) |g(x)| ≤ δ1 for a.e. x ∈ Q1 \ j Q2j , and P 2 1 (3) j |Qj | ≤ s|Q |. We can keep control of the cubes we are gathering and f . Indeed, clearly X X |Q2j | ≤ s |Q1k | ≤ s2 |Q0 |. j k And as for f , we notice that for a.e. x ∈ Q1 \ S j Q2j , |f (x)| ≤ |f (x) − mf (t, Q1 )| + |mf (t, Q1 | = |g(x)| + |mf (t, Q1 )| ≤ δ1 + δ0 + 10nβ0 ≤ (20n + 9)(β0 + β1 ). Continuing in this fashion, the computation becomes clear: Having selected the (k − 1)st generation of subcubes {Qk−1 }, we then select a kth generation of subcubes so that (1) with βj = 2φ(|Q0 |/2nj ) and δj = (10n + 9)βj , 0 ≤ j ≤ k − 1, we have P P P |f (x)| ≤ (10n + 9) k−1 δj + 10n k−2 βj ≤ (20n + 9) k−1 j=0 j=0 j=0 βj for S k k−1 a.e. x ∈ Q \ j Qj , and P k k (2) j |Qj | ≤ s |Q0 |. This is all that is needed. Suppose first that limu→0+ Ψ|Q0 | (u) = ∞. Then, for λ > 2(20n + 9)φ(|Q0 |), let k be the largest integer with the property that Pk−1 2(20n + 9) j=0 φ(|Q |/2nj ) < λ and observe that by (1) above {y ∈ Q0 : S k 0 |f (y)| > λ} ⊂ j Qj , and so by (2) above |{y ∈ Q0 : |f (y)| > λ}| ≤ sk |Q0 |. Medians, continuity, and vanishing oscillation 241 Furthermore, by this choice of k we have λ < 2(20n+9) k X j=0 2(20n + 9) φ(|Q0 |/2 ) ≤ n ln(2) 2n |Q  0| nj where c = 2(20n + 9)/n ln(2). So, |Q0 | −1 ≤ Ψ|Q (c2 λ), 0| 2kn Therefore,   du |Q0 | φ(u) = cΨ|Q0 | u 2kn kn |Q0 |/2 c2 = |{y ∈ Q0 : |f (y)| > λ}| ≤ sk |Q0 | ≤ n ln(2) . 2(4 + 10n) |Q0 | −1 ≤ Ψ|Q (c2 λ), 0| 2kn as we wanted to show. −1 And, for λ ≤ 2(20n + 9)φ(|Q0 |), pick c1 so that |Q0 | ≤ c1 Ψ|Q (c2 λ); since 0| {y ∈ Q0 : |f (y)| > λ} ⊂ Q0 , the conclusion holds. Clearly the argument works for all Q ⊂ Q0 . Finally, in case limu→0+ Ψ|Q0 | (u) < ∞, the above argument works for all integers k, and therefore f is an essentially bounded function on Q0 that satisfies (5.2). A more thorough argument shows that f is equivalent to an essentially Lipschitz function on Q0 [10]. It is now straightforward that if f ∈ vmos (Q0 ), then f ∈ VMO(Q0 ). Pick φ such that f ∈ bmos,φ,Q0 . Then by integrating (5.2) with respect to λ it follows that for all subcubes Q ⊂ Q0 , ∞   |f (y) − mf (1 − s, Q)| dy = |{y ∈ Q : |f (y) − mf (1 − s, Q)| > λ}| dλ 0 Q ≤ c1 ∞  −1 Ψ|Q| (c2 λ/kf ks,φ,Q0 ) dλ 0 ≤ ckf ks,φ,Q0 |Q|φ(2n |Q|). Therefore, 1  |f (y) − fQ | dy ≤ cφ(2n u) → 0 Q⊂Q0 , |Q|≤u |Q| sup Q as u → 0+ and f ∈ VMO(Q0 ). References [1] L. Carleson, BMO—10 years’ development, in: 18th Scandinavian Congress of Mathematicians (Aarhus, 1980), Progr. Math. 11, Birkhäuser, Boston, 1981, 3–21. 242 [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] J. Poelhuis and A. Torchinsky M. Cwikel, Y. Sagher, and P. Shvartsman, A new look at the John–Nirenberg and John–Strömberg theorems for BMO, J. Funct. Anal. 263 (2012), 129–166. N. Fujii, A condition for a two-weight norm inequality for singular integral operators, Studia Math. 98 (1991), 175–190. B. Jawerth and A. Torchinsky, Local sharp maximal functions, J. Approx. Theory 43 (1985), 231–270. F. John, Quasi-isometric mappings, in: Seminari 1962/63 Anal. Alg. Geom. e Topol., Vol. 2, Ist. Naz. Alta Mat., Ediz. Cremonese, Roma, 1965, 462–473. A. K. Lerner, A pointwise estimate for the local sharp maximal function with applications to singular integrals, Bull. London Math. Soc. 42 (2010), 843–856. A. K. Lerner, A simple proof of the A2 conjecture, Int. Math. Res. Notices 2012, doi: 10.1093/imrn/rns145. D. Sarason, Functions of vanishing mean oscillation, Trans. Amer. Math. Soc. 207 (1975), 391–405. X. L. Shi and A. Torchinsky, Functions of vanishing mean oscillation, Math. Nachr. 133 (1987), 289–296. S. Spanne, Some function spaces defined using the mean oscillation over cubes, Ann. Scuola Norm. Sup. Pisa (3) 19 (1965), 593–608. J.-O. Strömberg, Bounded mean oscillation with Orlicz norms and duality of Hardy spaces, Indiana Univ. Math. J. 28 (1979), 511–544. Jonathan Poelhuis, Alberto Torchinsky Department of Mathematics Indiana University Bloomington, IN 47405, U.S.A. E-mail: jpoelhui@umail.iu.edu torchins@indiana.edu Received July 11, 2012 (7565) 

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ZAKONODAVSTVO I PRAKSA UDK: 342.726-056.34(497.11) 364-54(497.11) doi: 10.5937/crimen1903278J Prihvaćeno: 08.12.2019. Miloš Janković* LIŠAVANJE SLOBODE U USTANOVAMA SOCIJALNE ZAŠTITE Apstrakt: Veliki broj korisnika u ustanovama socijalne zaštite domskog tipa su lica sa mentalnim invaliditetom, od kojih su mnogi zaključani ili na drugi način onemogućeni da svojevoljno napuste te ustanove. Faktički su lišeni slobode, uz objašnjenje da je to u njihovom „najboljem interesu“. Međutim, u pravnom sistemu Srbije ne postoji pravni osnov za lišavanje slobode korisnika u ustanovama socijalne zaštite. Takvo postupanje je u suprotnosti sa članom 27. Ustava Srbije po kome je „lišenje slobode dopušteno samo iz razloga i u postupku koji su predviđeni zakonom. To je u suprotnosti i sa članom 5. Evropske konvencije o ljudskim pravima po kome „niko ne može biti lišen slobode osim iz razloga i u skladu sa zakonom propisanim postupkom“. Nakon višegodišnjeg ignorisanja preporuka Zaštitnika građana da se prestane sa lišavanjem slobode koje nema valjan pravni osnov, Ministarstvo nadležno za socijalna pitanja je tokom 2019. godine pokušalo da navedeno postupanje uredi zakonom. Međutim, ponuđene verzije Nacrta zakona sadrže brojne nedostatke i u suprotnosti su sa važećim standardima. Ovaj rad predstavlja pokušaj da se argumentovano ukaže na neodrživost ponuđenih rešenja. Ključne reči: lišenje slobode, socijalna zaštita, ustanove, prinudni smeštaj, mentalne smetnje, tortura, sputavanje UVOD U ustanovama socijalne zaštite je prema dostupnim podacima smešteno oko 18.000 korisnika1. Značajan broj njih su lica sa mentalnim smetnjama i osobe sa smetnjama u razvoju. Ta lica su uglavnom zaključana ili su na drugi način onemogućena da svojevoljno napuste ustanove, odnosno faktički su lišeni slobode, uz objašnjenje da je to u njihovom „najboljem interesu“. Imajući u vidu da u pravnom sistemu Republike Srbije ne postoji pravni osnov za prinudno smeštanje, odnosno lišavanje slobode korisnika u ustanovama socijalne zaštite, takvo postupanje je u suprotnosti sa odredbom iz člana 20. Ustava Republike * 1 advokat, milosr.jankovic@gmail.com . B. Janjić, D. Ćirić Milovanović /2017/: Ovde i zidovi imaju uši, Beograd, Inicijativa za prava osoba sa mentalnim invaliditetom MDRI-S. Miloš Janković, Lišavanje slobode u ustanovama socijalne zaštite 279 Srbije2 po kome se „ljudska prava zajemčena Ustavom mogu zakonom ograničiti samo ako ograničenje dopušta Ustav“ i člana 27. Ustava po kome je „lišenje slobode dopušteno samo iz razloga i u postupku koji su predviđeni zakonom”. Osim toga, ono je u suprotnosti i sa članom 5. Evropske konvencije o ljudskim pravima po kome „niko ne može biti lišen slobode osim iz razloga i u skladu sa zakonom propisanim postupkom“. Nakon višegodišnjeg ignorisanja preporuka Zaštitnika građana da se prestane sa lišavanjem slobode koje nema valjan pravni osnov, Ministarstvo nadležno za socijalna pitanja je tokom 2019. godine pokušalo da navedeno postupanje uredi zakonom. Međutim, ponuđene verzije Nacrta zakona sadrže brojne nedostatke i u suprotnosti su sa važećim standardima. Ovaj rad predstavlja pokušaj da se argumentovano ukaže na neodrživost ponuđenih rešenja. Analiza izložena u ovom radu zasnovana je na neposrednim saznanjima stečenim tokom desetogodišnjeg ostvarivanja mandata Zaštitnika građana u oblasti zaštite prava lica lišenih slobode i višegodišnjeg obavljanja poslova Nacionalnog mehanizma za prevenciju torture (NPM). Na osnovu propisanih ovlašćenja i preuzetih dužnosti, autor ovog rada, kao nadležni zamenik Zaštitnika građana, kontrolisao je zakonitost i pravilnost postupanja prema licima lišenim slobode. U svojstvu rukovodioca NPM kontinuirano je obavljao posete najrazličitijim mestima u kojima se nalaze lica lišena slobode. Osim što je posećivao ustanove klasične detencije poput policijskih stanica, pritvora i zatvora, posećivao je i one netipične, kao što su psihijatrijske ustanove i ustanove socijalne zaštite domskog tipa. Cilj je bio utvrđenje da li je postupanje prema licima lišenim slobode usklađeno sa važećim nacionalnim propisima, standardima i pravilima međunarodnog prava na čije sprovođenje se Srbija obavezala. RELEVANTNI POJMOVI Lišenje slobode Na početku, ukazujemo Ustavom Republike Srbije, u članu 27. propisano je da „svako ima pravo na ličnu slobodu” i da je „lišenje slobode dopušteno samo iz razloga i u postupku koji su predviđeni zakonom”, a u članu 25. da „niko ne može biti izložen mučenju, nečovečnom ili ponižavajućem postupanju ili kažnjavanju“. Navedene odredbe Ustava predstvaljaju dosledno otelotvorenje odredbi najznačajnijih međunarodnih dokumenata kao što su Univerzalna deklaracija o ljudskim pravima3, Pakt o političkim i građanskim pravima4 i Evropska konvencija o ljudskim pravima5. U tom smislu je odredba iz člana 14. Konvencije o pravima osoba sa in2 3 4 5 „Službeni glasnik RS“, br. 98/2006. https://www.ohchr.org/EN/UDHR/Documents/UDHR_Translations/cnr.pdf, 05.decembar 2019. Zakon o ratifikaciji Međunarodnog pakta o građanskim i političkim pravima („Službeni list SFRJ“, br. 7/1971–73). Zakon o ratifikaciji Evropske konvencije za zaštitu ljudskih prava i osnovnih sloboda („Službeni list SCG – Međunarodni ugovori“, br. 9/2003 i 5/2005). 280 CRIMEN (X) 3/2019 • str. 278–298 validitetom6 kojom je propisano da „osobe sa invaliditetom ravnopravno sa drugima: (a) uživaju pravo na ličnu slobodu i bezbednost; (b) ne budu lišena slobode protivzakonito ili nečijom samovoljom, da svako lišavanje slobode bude u skladu sa zakonom, kao i da postojanje invaliditeta ni u kom slučaju ne može biti opravdanje za lišavanje slobode. Svesni postojanja brojnih nedoumica o tome šta predstavlja lišenje slobode, ukazaćemo na određenje tog pojma kako je to navedeno članu 4. Opcionog protokola uz Konvenciju protiv torture i drugih surovih, neljudskih ili ponižavajućih kazni ili postupaka7 (u daljem tekstu: Opcioni protokol), te ćemo u daljem tekstu pod lišenjem slobode, odnosno detencijom podrazumevati „bilo koji oblik zatvaranja ili stavljanja lica u javno ili privatno mesto čuvanja, koje to lice prema odluci sudskog, upravnog ili drugog tela ne može svojevoljno da napusti”. Shodno navedenoj odredbi Opcionog protokola, mestom detencije smatra se „svako mesto pod nadležnošću i upravom (kontrolom) države u kome se nalaze lica koja su lišena slobode, bilo po osnovu naredbe izdate od strane državnog organa (organa javne vlasti) ili na njegov podsticaj, ili uz njenu (njegovu) saglasnost ili pristanak”. Na osnovu napred predočenog, možemo zaključiti da je mesto detencije svako mesto kojem je to svojstvo utvrđeno propisima, ali i bilo koje drugo mesto u kome se faktički vrši lišavanje slobode, bez obzira što mu to svojstvo nije utvrđeno propisima. Da bi se određeno mesto smatralo mestom detencije dovoljno je da se u njemu, bilo po osnovu naredbe izdate od strane organa javne vlasti ili na njegov podsticaj, ili uz njegovu saglasnost ili pristanak, nalaze ili se mogu nalaziti lica koje ga ne mogu samovoljno napustiti. Mesta koja shodno propisima Republike Srbije predstvljaju mesta detencije su npr. policijske stanice (u kojima se izvršava mera zadržavanja), zavodi za izvršenje krivičnih sankcija (u kojima se izvršava mera pritvora, kazna zatvora i mera bezbednosti obaveznog psihijatrijskog lečenja i čuvanja), kao i psihijatrijske ustanove (u kojima se vrši zadržavanje i smeštaj bez pristanka lica sa mentalnim smetnjama). Mesta kojima propisima Republike Srbije nije utvrđeno svojstvo mesta detencije, ali u kojima se potem prinudnog smeštaja vrši lišavanje slobode su brojne socijalne ustanove domskog tipa. To su državne ili privatne ustanove koje su pod kontrolom državnih organa, a u kojima je veliki broj korisnika smešten mimo njihove volje, odnosno koje ne mogu samovoljno da napuste. Osobenost ustanova detencije je da su dešavanja u njima sakrivena od očiju javnosti. U njima se sučeljavaju službena lica (policijski i zatvorski službenici, medicinsko osoblje i osoblje ustanova socijalne zaštite) nasuprot pojedincu koji je potpuno zavistan od njihovog odnosa prema njemu, što je plodno tlo za nastanak zlostavljanja, pa i torture. Lica u detenciji, naročito ako su lica sa mentalnim smet6 7 Zakon o ratifikaciji Konvencije o pravima osoba sa invaliditetom („Sl. glasnik RS – Međunarodni ugovori“, br. 42/2009). Zakon o ratifikaciji Opcionog protokola uz Konvenciju UN protiv torture i drugih surovih, neljudskih ili ponižavajućih kazni ili postupaka („Službeni list SCG – Međunarodni ugovori“, br. 16/2005 i 2/2006). Miloš Janković, Lišavanje slobode u ustanovama socijalne zaštite 281 njama ili smetnjama u razvoju, kontinuirano su izložena visokom riziku. Osim toga, „neodgovarajući materijalni uslovi smeštaja i pojedina postupanja nadležnih organa prema licima lišenim slobode koja su rezultat nedostataka u sistemu i predstavljaju lošu praksu koja nije u skladu sa važećim standardima, imajući u vidu njihov intenzitet i trajanje, mogu prerasti, a u pojedinim slučajevima i prerastaju, u ponižavajuće ili nečovečno postupanje, pa i torturu”8. 1.2. Osobe sa mentalnim smetnjama Postojeći Zakon o zaštiti lica sa mentalnim smetnjama9 značajan je što je, nakon dugog niza godina priprema, njime po prvi put regulisana ova oblast, čime je načinjen veliki korak napred u pogledu zaštite prava lica sa mentalnim smetnjama, a time i onih koji su hospitalizovani u psihijatrijskim bolnicama. Iako je Zakon u velikoj meri usklađen sa važećim međunarodnim standardima, neophodno je njegovo značajno unapređenje.10 Nedostaci Zakona se naročito odnose na „stvaranje uslova za što kraće zadržavanje pacijenata sa mentalnim smetnjama u psihijatrijskim ustanovama, pre svega u velikim psihijatrijskim bolnicama. U tom cilju, neophodno je izvršiti izmene i dopune Zakona radi stvaranja efikasnog i održivog sistema „deinstitucionalizacije“, koja osim zdravstvenog, između ostalog, u velikoj meri podrazumeva i socijalni aspekt. Potrebno je propisati odgovarajuće uslove za zbrinjavanje, odnosno svakodobnu i svakovrsnu podršku osobama sa mentalnim smetnjama (uključujući i njihove porodice) za život van psihijatrijskih ustanova, u okruženju koje je u što manjoj meri određeno njihovim mentalnim smetnjama”11. Za potrebe ovog rada posebno treba ukazati da je u tački 1, stav 1, član 2 Zakona navedeno da je lice sa mentalnim smetnjama „nedovoljno mentalno razvijeno lice, lice sa poremećajima mentalnog zdravlja, odnosno lice obolelo od bolesti zavisnosti”. Navedeno određenje je potpuno nejasno, ostalo je bez bilo kakvog objašnjenja šta znači „poremećaj mentalnog zdravlja”, a nije ni određena granica koja čini distinkciju između dovoljnog i „nedovoljnog” mentalnog razvoja određenog lica. Uvažavajući praksu Evropskog suda za ljudska prva i referentne taksonomije, pre svega važeću klasifikaciju Svetske zdravstvene organizacije (ICD 1012), pod licima sa mentalnim smetnjama mogli bi podrazumevati obolele od hroničnog psihotičnog poremećaja – shizofrenije, shizotipskih poremećaja i sumanutih poremećaja iz grupe F20-F2913, kao i drugih mentalnih poremećaja koji zahtevaju intenzivan psihijatrijski tretman14. 8 9 10 11 12 13 14 M. Janković /2018/: Položaj osoba sa mentalnim smetnjama lišenih slobode, Sociologija Vol. LX, n° 4. „Službeni glasnik RS“, br. 45/2013. Videti više: M. Janković /2015/: Slobode i prava lica sa mentalnim smetnjama – nedostaci Zakona o zaštiti lica sa mentalnim smetnjama, Crimen (VI) 1, pp. 61–80. Izveštaj NPM za 2017. godinu, p 7. Svetska zdravstvena organizacija /2010/: Deseta revizija Međunarodne statističke klasifikacije bolesti i srodnih zdravstvenih problema, pp. 311–316. SZO: ibid. Zaštitnik građana, Mišljenje 12–3077/13, fn. 1. 282 CRIMEN (X) 3/2019 • str. 278–298 U tom pogledu, ostaju otvorena pitanja, poput toga da li su lica koja imaju poteškoće u učenju ili dementna lica uključena u krug osoba koje Zakon određuje kao lica sa mentalnim smetnjama, a ako jesu, u koje svrhe, posebno da li su obuhvaćeni i u pogledu Zakonom propisane detencije i sputavanja. Nepostojanje bilo kakvog određenja šta Zakon podrazumeva pod „nedovoljno mentalno razvijenim licem”, odnosno „licem sa poremećajima mentalnog zdravlja”, u praksi može dovesti, pa i dovodi, do nejednake primene propisa, pa i mogućih zloupotreba. Napred navedeni nedostatak Zakona još više dobija na značaju imajući u vidu da se i stručnjaci psihijatrije često suočavaju sa „pitanjem granice između zdravog i bolesnog, naročito granice koju određuju prisustvo, odnosno odsustvo mentalnih smetnji, a koje su nekada vrlo zamagljene”15. Naime, „opšteprihvaćeni medicinski model bolesti koji postavlja krutu distinkciju zdravlja od mentalne bolesti, stvarajući dihotomiju u smislu da je osoba ili bolesna ili zdrava, ne uvažava činjenicu da su mnogi simptomi i znaci mentalnih poremećaja deo svakodnevnog životnog iskustva, odnosno da se sreću kod osoba koje se označavaju „zdravim“.16 Dalje, potrebno je navesti podatak Svetske zdravstvene organizacije da je nа osnovu sistemske analize opšte populacije u većini evropskih zemalja pokazano je da će 27% odrasle populacije starosti 18–65 godina nekada tokom svog života imati mentalni poremećaj.17 Imajući u vidu napred navedeno, mogućnost arbitrarnosti pa i zloupotreba u praksi naročito se ogleda u slučajevima detencije kada se shodno članu 20. Zakona vrši smeštanje u psihijatrijsku ustanovu lica sa mentalnim smetnjama na zahtev i uz pismeni pristanak njegovog člana uže porodice ili njegovog zakonskog zastupnika, kao i kada se shodno članu 21. Zakona, po postupku za zadržavanje bez pristanka i smeštaj bez pristanka, vrši smeštanje u psihijatrijsku ustanovu lica sa mentalnim smetnjama za koje se proceni da, usled mentalnih smetnji, ozbiljno i direktno ugrožava sopstveni život ili zdravlje ili bezbednost, odnosno život ili zdravlje ili bezbednost drugog lica. Očigledno je da je neophodno unaprediti odredbe Zakona koje se odnose na detenciju i sputavanje, na taj način što će se propisati nedostajući mehanizami zaštite onih čija se prava ograničavaju, a koji moraju biti u skladu sa važećim standardima. Pre svega, da bi neko bio lišen slobode nužno je da se zakonom propišu precizni, jasni i nesumnjivi uslovi koji moraju biti ispunjeni. Dalje, neophodno je da se detaljno propiše procedura koja mora biti sprovedena, tako da se u postupku omogući učešće onoga o čijoj slobodi i pravima se odlučuje bez obzira na njegove mentalne kapacitete, da mu se o trošku države omogući pravna pomoć, da nakon neposrednog suočavanja sudije sa tim licem, na osnovu stručnog mišljenja nezavisnog psihijatra konačnu odluku o lišavanju slobode donese sud, da postoji mogućnost žalbe na tu odluku, kao i obaveza da se u shodnoj proceduri vrši njeno periodično preispitivanje u razumnim rokovima. 15 16 17 V. Jović /2015/: Opšta psihopatologija sa konceptualnom istorijom, K. Mitrovica, Filozofski fakultet Univerziteta u Prištini, p. 6. D. Kecmanović /2008/: Psihijatrija u kritičkom ogledalu, Beograd, Službeni glasnik. http://www.euro.who.int/en/health-topics/noncoHYPERLINK, 29. novembar 2019. Miloš Janković, Lišavanje slobode u ustanovama socijalne zaštite 283 U vezi postizanja viših standarda u oblasti sputavanja, naročito ako se ono vrši vezivanjem, odgovor je moguće naći u brojnim preporukama Zaštitnika građana kojima je ukazano na sve elemente koje je je potrebno ispuniti da bi postupak sputavanja bio u skladu sa važećim standardima i praksom relevantnih međunarodnih tela.18 Socijalna zaštita Socijalna zaštita, prema članu 2. važećeg Zakona o socijalnoj zaštiti19 je „organizovana društvena delatnost od javnog interesa čiji je cilj pružanje pomoći i osnaživanje za samostalan i produktivan život u društvu pojedinaca i porodica, kao i sprečavanje nastajanja i otklanjanje posledica socijalne isključenosti”. Članom 5. Zakona propisano je da usluge socijalne zaštite čine „aktivnosti pružanja podrške i pomoći pojedincu i porodici (u daljem tekstu: korisnik) radi poboljšanja, odnosno očuvanja kvaliteta života, otklanjanja ili ublažavanja rizika nepovoljnih životnih okolnosti, kao i stvaranje mogućnosti da samostalno žive u društvu. Shodno tome, možemo zaključiti da su korisnici usluga socijalne zaštite lica koja dobijaju podršku i pomoć radi poboljšanja, odnosno očuvanja kvaliteta njihovih života, otklanjanja ili ublažavanja rizika nepovoljnih životnih okolnosti, kao i radi stvaranja mogućnosti da samostalno žive u društvu. Nasuprot elementima i ciljevima socijalne zaštite iz početnih odredbi Zakona kojima se naglašava uloga socijalne zaštite u stvaranju uslova za život u društvu i sprečavanju socijalne isključenosti, u daljem tekstu Zakona značajan broj odredbi je posvećen institucionalnom zbrinjavanju korisnika socijalne zaštite. Tako je članom 40. Zakona propisano da usluge socijalne zaštite čine i „usluge smeštaja”, između ostalog i „domski smeštaj”, a članom 60. Zakona je propisana mogućnost stvaranja „socijalno-zdravstvenih” ustanova, odnosno posebnih organizacionih jedinica u okviru ustanova socijalne zaštite ili zdravstvenih ustanova. Pažnju zavređuje član 26. Zakona kojim je propisano da se usluge socijalne zaštite pružaju u skladu sa „najboljim interesom korisnika”. Kada su u pitanju osobe sa mentalnim invaliditetom, shodno stavovima iz Opšteg komentara br. 1 Komiteta za prava osoba sa invaliditetom iz 2014. godine20, to predstavlja zadržavanje prevaziđenog režima „zamenskog odlučivanja“, po kome se osobi sa mentalnim invaliditetom oduzima mogućnost da shodno sopstvenoj volji (želji, htenju) donosi odluke koje se nje tiču, nameće joj se neko drugi ko donosi odluke umesto nje, uključujući i suprotno njenoj volji, tako da je odluka druge osobe zasnovana na onome što se smatra objektivnim „najboljim interesom“, a ne na autentičnoj volji i sklonostima određene osobe.21 Stav Komiteta je da zamensko odlučivanje treba zameniti „paradigmom podrške odlučivanju (potpomognuto odlučivanje)“. 18 19 20 21 Zaštitnik građana, Preporuka Opštoj bolnici u Šapcu 61 – 1885 / 14. „Službeni glasnik RS“, br. 24/2011. http://www.mdri-s.org/wp-content/uploads/2015/02/Mucenje-kao-lecenje.pdf N. Mole, F. Auer i B. Braithwaite /2014/: Međunarodni i uporedni standardi – in: Prava osoba sa mentalnim invaliditetom (S. Gajin, ed.), Beograd, Centar za unapređenje pravnih studija. 284 CRIMEN (X) 3/2019 • str. 278–298 Za potrebe ovog rada posebno treba ukazati da ni jedna odredba Zakona, niti drugi propis istog ranga, ne propisuje bilo koji oblik prinudnog smeštanja, odnosno lišavanja slobode korisnika u ustanovama socijalne zaštite. Imajući u vidu da odredba člana 27. Ustava jasno propisuje da je „lišenje slobode dopušteno samo iz razloga i u postupku koji su predviđeni zakonom, možemo zaključiti da u Srbiji ne postoji valjan pravni osnov za lišavanje slobode korisnika ustanova socijalne zaštite, odnosno da je u tim ustanovama detencija nezakonita. Napred navedeno se podjednako odnosi i na bilo koji oblik sputavanja korisnika u ustanovama socijalne zaštite, bilo da je reč o fizičkom sputavanju (ručno sputavanje ili vezivanje) ili hemijskom sputavanju. POLOŽAJ KORISNIKA U USTANOVAMA SOCIJALNE ZAŠTITE DOMSKOG TIPA 2.1. Nalazi civilnog društva Kada je u pitanju položaj korisnika u ustanovama socijalne zaštite u Srbiji, jedan od najznačajnijih dokumenata je izveštaj nevladine organizacije Mental Disability Rights International (MDRI), objavljen 2007. godine22, u kome je prvi put u Srbiji na ilustrativan način ukazano da se u ustanovama socijalne zaštite domskog tipa, u konkretnom slučaju u ustanovi socijalne zaštite u Kulinama, postupanje prema korisnicima može okarakterisati kao zlostavljanje. Navedeni izveštaj je razotkrio nešto što je decenijama čuvano sakriveno od očiju javnosti, i od strane nadležnih prikazivano kao zbrinjavanje korisnika uz poštovanje njihovog „najboljeg interesa”. Lavina osuda koja se obrušila na autore tog izveštaja od strane predstavnika vlasti, ali i osoblja tih ustanova, bez iznošenja valjanih argumenata, samo je uverila javnost u osnovanost iznetih kvalifikacija i provocirala ostale branitelje ljudskih prava da počnu intezivnije da prate položaj korisnika u ustanovama socijalne zaštite. 2.2. Nalazi Zaštitnika građana Ostvarujući svoj mandat, a u velikoj meri podstaknut napred navedenim izveštajem MDRI, Zaštitnik građana je u prvih deset godina svog rada posvetio pažnju, između ostalog, i položaju lica koja su prinudno smeštana u ustanovama socijalne zaštite. Aktivnosti zaštitnika građana shodno mandatu određenom Zakonom o zaštitniku građana23 nisu bile brojne, pre svega iz razloga malobrojnih pritužbi. Naime korisnici u ustanovama socijalne zaštite još nisu osnaženi (za verovati i sputani od strane osoblja ustanova) da se bore za svoja prava poput drugih lica lišenih slobode, na primer zatvorenika. Znatno intezivnije aktivnosti Zaštitnik je ostvario obavljajući poslove NPM u cilju prevencije zlostavljanja korisnika u tim ustanovama. U saradnji sa nevladinim organizacijama (pre svega sa MDRI-S) i spoljnim ekspertima (većinom lekarima forenzičarima, psihijatrima i psiholozima) obavljen je značajan broj poseta ustanovama socijalne zaštite. Po obavljenoj poseti sačinjavani 22 23 http://www.mdri-s.org/wp-content/uploads/2015/02/Mucenje-kao-lecenje.pdf. „Službeni glasnik RS“, br. 79/2005 и 54/2007. Miloš Janković, Lišavanje slobode u ustanovama socijalne zaštite 285 se izveštaji koji sadrže informacije o uočenom stanju, utvrđenim nedostacima, kao i preporuke za unapređenje zaštite od torture i zlostavljanja24. Kao suštinski zaključak koji se može uočiti iz tih izveštaja je da je veliki broj korisnika smešten u ustanovama socijalne zaštite koje ne mogu da svojevoljno napuste, i da je osnovni razlog to što nisu uspostavljeni uslovi za njihov život u zajednici. Očigledno je da se u Srbiji ne ostvaruje proklamovana deinstitucionalizacija, već da je imanentno izopštavanje osoba sa invaliditetom u socijalne ustanove koje poprimaju azilni karakter, s tim da je među njima veliki broj osoba sa mentalnim smetnjama i smetnjama u razvoju, kojima je neophodno omogućiti život uz podršku van institucija. Osobama sa mentalnim smetnjama je prevashodno potrebno obezbediti lečenje, i to psihijatrijsko vanbolničko lečenje, a izuzetno i na kratak rok lečenje u prsihijatrijskoj ustanovi, u svakom slučaju ne u ustanovama socijalne zaštite. Zaštitnik građana je ispravno ukazao da se višegodišnje „lečenje“, u mnogim slučajevima „doživotno“ nikako ne može smatrati lečenjem. Izveštaji NPM su posebnu pažnju posvetili smeštajnim uslovima. Ukazano je da se većina ustanova socijalne zaštite domskog tipa nalazi van naseljenih mesta, tako da je korisnicima znatno otežano ostvarivanje interakcije sa lokalnom zajednicom, što ima za posledicu da su im sve životne aktivnosti koncentrisane unutar izolovane ustanove25. Dalje, utvrđeno je da je veliki broj korisnika smešten u ustanovama koje su veoma udaljene od prebivališta korisnika, što im onemogućuje ostvarivanje intezivnijeg kontakta sa njihovim porodicama i drugim bliskim licima. Uočeni su i brojni nedostaci kvaliteta smeštaja, pojedini kapaciteti su stari, neodržavni, a pojedini su prenaseljeni. Nedostatak nege korisnika u velikoj meri je posledica nedostatka negovatelja, a kada je u pitanju zdravstvena zaštita primetan je nedovoljan broj psihijatara. Iz izveštaja NPM nesumnjivo proističe da se u ustanovama socijalne zaštite domskog tipa vrši lišavanje slobode korisnika. Naime, veliki broj korisnika je zatečen zaključan u objektima ustanova, na koji način su fizički onemogućeni da ih svojevoljno napuste. Pojedini korisnici su na dug rok bili smešteni u prostorijama pod rešetkama, nalik na zatvorske ćelije a neke od njih poput kaveza.26 Osoblje ustanova domskog tipa je dugo vremena negiralo da su njihovi korisnici lišeni slobode, a činjenicu da su zaključani, opravdavali su time da se sve, pa i to čini u „najboljem interesu korisnika“. Tvrdili su da su njihovi korisnici dobrovoljno smešteni i da mogu da napuste ustanovu kada god hoće. Takvi navodi su gubili smisao nakon što bi na postavljeno pitanje šta bi se desilo kada bi korisnik otišao iz institucije, prisutno osoblje odgovorilo da bi se po redovnoj proceduri pozvala policija da ga vrati u ustanovu. Ukazano je i na pojavu fizičkog sputavanja korisnika na dug rok, najčešće vezivanjem, i to uglavnom nestandardnim sredstvima (običnim kaiševima, čaršavima, zavojima i sl.). Naglašeno je i da je uočeno da se pojedini korisnici na dug rok drže u izolaciji, kao i da je pojedinim korisnicima to redovni smeštaj. Po objavljivanju izveštaja o poseti ustanovi socijalne zaštite Veternik, u kome su konstatovani i fo24 25 26 Videti više u publikaciji Saveta Evrope https://npm.rs/attachments/article/850/Zastitnik%20gradjana%20o%20 osobama%20sa%20mentalnim%20smetnjama.pdf, 04. decembar 2019. Videti više u Godišnjem izveštaju Zaštitnika građana i NPM za 2017. godinu. Izveštaj Zaštitnika građana o poseti domu Veternik, br. 71 –26/ 13. 286 CRIMEN (X) 3/2019 • str. 278–298 tografijama argumentovani slučajevi dugoročne izolacije korisnika u prostoriji pod rešetkama, nadležni ministar je u javnom nastupu takvo postupanje pokušao da opravda tvrdnjom da su ta lica „kanibali”. 2.3. Nalazi organa vlasti Vlada Republike Srbije u Strategiji za zaštitu mentalnog zdravlja u Srbiji za period 2019–2026., navodi da „u velikim ustanovama socijalne zaštite (sa domskim smeštajem) su dugotrajno (više godina) smeštene osobe sa hroničnim psihozama i poremećajem intelektualnog razvoja. Slično kao i u psihijatrijskim bolnicama i u velikim ustanovama socijalne zaštite često su loši materijalni uslovi, a broj osoblja je mali u odnosu na broj korisnika”27, i konstatuje da je u Srbiji „odsustvo centara za zaštitu mentalnog zdravlja u lokalnoj zajednici i drugih nestacionarnih psihijatrijskih službi.”28 POKUŠAJI ZAKONSKOG REGULISANJA LIŠAVANJA SLOBODE U USTANOVAMA SOCIJALNE ZAŠTITE Nakon višegodišnjeg oglušivanja na ukazivanje da se prinudno smeštanje, odnosno lišavanje slobode, kao i sputavanje u ustanovama socijalne zaštite vrši nezakonito, donosioci odluka u Srbiji su napokon prihvatili da je neodrživo dalje prinudno smeštanje i sputavanje korisnika u ustanovama socijalne zaštite uz puko opravdavanje da se ono vrši na osnovu mišljenja stručnih službi da je to u „najboljem interesu korisnika”. Ministarstvo nadležno za socijalna pitanja29 je rešenje napred navedenog problema videlo u donošenju zakona kojim će se ustanoviti pravni osnov za takvo postupanje. Shodno tome, početkom 2019. godine javnosti je predočena prva verzija Nacrta zakona, naslovljena „Zakon o zaštiti lica sa mentalnim smetnjama u socijalnoj zaštiti”, a krajem iste godine svetlost dana je ugledala i druga verzija, naslovljena „Zakon o zaštiti prava korisnika usluga smeštaja u socijalnoj zaštiti”. 3.1. Prva verzija Nacrta zakona Prvi tekst nacrta kojim je pokušano da se stvori prvni osnov za lišavanje slobode i sputavanje korisnika u ustanovama socijalne zaštite učinjen je pod plaštom navodne ideje o potpunijoj zaštiti osoba sa mentalnim smetnjama i naslovljen je „Zakon o zaštiti lica sa mentalnim smetnjama u socijalnoj zaštiti”. Autor ovog članka je na javnoj raspravi održanoj u maju 2019. godine u Beogradu izneo niz suštinskih nedostataka tog Nacrta zakona. Značajno je napomenuti da je, osim prisutnih predstavnika civilnog društva, iznete prigovore izričito podržao i prisutni predsednik Nacionalne komisije za mentalno zdravlje, ukazavši 27 28 29 Strategija za zaštitu mentalnog zdravlja u Srbiji za period 2019–2026, p. 7. Ibid., p. 8. Ministarstvo za rad, zapošljavanje, boračka i socijalna pitanja. Miloš Janković, Lišavanje slobode u ustanovama socijalne zaštite 287 da u procesu izrade zakona uopšte nije konsultovano Ministarstvo zdravlja Srbije, niti Nacionalna komisija. U tom kontekstu zabrinjavajuće su delovala i javno izneta uveravanja predstavnika Ministarstva nadležnog za socijalna pitanja da će Zakon biti zvaničan predlog Vlade Srbije i do kraja godine usvojen u Narodnoj skupštini u svakom slučaju, bez obzira na ukazane suštinske primedbe. Osnovna primedba na Nacrt zakona odnosila se na to da u srpskom pravnom sistemu već postoji Zakon o zaštiti osoba sa mentalnim smetnjama. Iznet je stav da je neodrživo donošenje novih zakona koji štite prava pripadnika određene povredive grupe zavisno od vrste institucije u kojima su ta lica smeštena. Imajući u vidu potpuno odsustvo saradnje relevantnih ministarstava i činjenicu da Ministarstvo zdravlja i Nacionalna komisija za mentalno zdravlje nisu učestvovali u izradi Nacrta zakona, možemo zaključiti da cilj Ministarstva nadležnog socijalnu politiku nije bila zaštita osoba sa mentalnim smetnjama, već zakonsko ojačavanje ustanova nad kojima je nadležano i ozakonjenje prakse lišavanja slobode koje ustanove za socijalnu zaštitu decenijama nezakonito sprovode. Ukazano je da umesto takvog partikularnog pristupa, srpske vlasti treba da se posvete opštoj zaštiti ranjivih osoba kao što su osobe sa mentalnim invaliditetom, posebno u vezi sa kontroverzom njihovog dugoročnog lišavanja slobode u zatvorenim ustanovama bez obzira na vrstu ustanove i koje ministarstvo je nadležno nad njima. U tom smislu, u Strategiji za zaštitu mentalnog zdravlja u Srbiji za period 2019–2026. je navedeno „mentalno zdravlje se tiče svih i zahteva partnersku akciju relevantnih ministarstava, stručnjaka i udruženja korisnika i pružalaca usluga”. Naglašeno je da prisilno smeštanje osoba sa mentalnim invaliditetom u zatvorene ustanove, posebno na duži rok, treba da bude izuzetak. Ako je njihovo obavezno smeštanje neophodno, ono mora biti poslednja mera, na što kraći rok i u specijalizovanim zdravstvenim ustanovama u kojima im je moguće obezbediti adekvatno lečenje. Otvoreno je i suštinsko pitanje opravdanosti prisilnog smeštanja, odnosno lišavanja slobode osoba sa mentalnim smetnjama u ustanovama socijalne zaštite. Ukazano je da je sporno da li karakter tih ustanova, nadležnih da pružaju usluge socijalne zaštite svojim korisnicima, dozvoljava da se u njima vrši lišavanje slobode. Postavljena je kao upitna konstrukcija da lišavanje slobode predstavlja „uslugu korisniku socijalne zaštite”. Osim navedenih, iznete su i druge brojne primedbe. 3.2. Druga verzija Nacrta zakona Radna grupa za izradu Nacrta zakona, verovatno svesna opravdanosti brojnih iznetih primedbi, pristupila je sačinjavanju druge verzije Nacrta zakona. Međutim, očigledno pritisnuti zahtevom da se donese propis kojim će se legalizovati dosadašnje nezakonito prinudno smeštanje, odnosno lišavanje slobode velikog broja korisnika u ustanovama socijalne zaštite, nisu učinjene suštinske izmene. U drugoj verziji Nacrta zakona, koja je ugledala svetlost dana krajem 2019. godine, veliki broj primedbi iznetih tokom javne rasprave na prvu verziju Nacrta zakona nije prihvaćen. 288 CRIMEN (X) 3/2019 • str. 278–298 Na početku, ukazao bih da je radna grupa prihvatila primedbu na prvi tekst Nacrta zakona da je potpuno neodgovrajući njegov naziv „Zakon o zaštiti lica sa mentalnim smetnjama u socijalnoj zaštiti”. Izvršena je izmena, te je u drugom Nacrtu zakona isti naslovljen sa „Zakon o zaštiti prava korisnika usluga smeštaja u socijalnoj zaštiti“. Shodno primedbi da nije odgovarajuće da predmet zakona bude zaštita lica sa mentalnim smetnjama, jer takav zakon već postoji, pisci Nacrta zakona su, uglavnom zadržavši postojeću sadržinu teksta prvog Nacrta, iz druge verzije Nacrta zakona brisali izraz „osobe sa mentalnim smetnjama“, tako da se u drugom Nacrtu ne spominju (osim na nekoliko mesta, u nereferentnom kontekstu po ovom pitanju). Iz navedenog proističe da je za razliku od prvog Nacrta zakona, čiji je predmet bio zaštita osoba sa mentalnim smetnjama, predmet druge verzije Nacrta zakona zaštita svih korisnika u ustanovama socijalne zaštite. Imajući u vidu da svi korisnici usluga u ustanovama socijalne zaštite nisu osobe sa mentalnim smetnjama, odnosno da korisnici predstavljaju daleko širi krug lica od osoba sa mentalnim smetnjama, postavlja se pitanje kako tekst druge verzije Nacrta zakona, koji je minimalno modifikovan (brisanjem izraza „osobe sa mentalnim smetnjama”) može imati drugačiji, odnosno daleko širi predmet zaštite i odnositi se na sve korisnike. Navedeno potvrđuje osnovanost napred istaknutog prigovora da piscima Nacrta nije bitan predmet zaštite, odnosno zaštita prava lica koj su smeštena u ustanovama socijalne zaštite, bilo da se radi o osobama sa mentalnim smetnjama ili o svim korisnicima. Očigledna je intencija da se zaštite institucije, legalizuje postojeća praksa postupanja prema korisnicima i stvori pravni osnov za nastavak takve prakse i ubuduće. Nezavisno od promenjenog naziva zakona u drugoj verziji Nacrta zakona, kao i brisanja „osoba sa mentalnim smetnjama” iz njegovog teksta, imajući u vidu da se ta verzija odnosi na sve korisnike u ustanovama socijalne zaštite, time i na korisnike sa mentalnim smetnjama, sve konsekvence zakona odnose se i na njih. Međutim, okolnost da se druga verzija Nacrta odnosi na sve korisnike u ustanovama socijalne zaštite ne znači da više ne stoje primedbe koje su prethodno bile iznete na prvi Nacrt zakona, a odnose se na lišavanje slobode i sputavanje. KOMENTARI POJEDINIH REŠENJA U NACRTU ZAKONA Uzevši u obzir poslednju dostupnu verziju Nacrta zakona, u daljem tekstu ukazaću na uočene nedostatke i dileme koje otvaraju postojeća rešenja. 4.1. Paradigma najboljeg interesa korisnika U Nacrtu zakona je paradigma „najboljeg interesa korisnika“ postavljena kao jedno od osnovnih načela. Propisano je da se „usluge smeštaja pružaju u skladu sa najboljim interesom korisnika“. Kada su u pitanju osobe sa mentalnim invaliditetom, shodno stavovima iz Opšteg komentara br. 1 Komiteta za prava osoba sa invaliditetom iz 2014. godine30, to predstavlja zadržavanje prevaziđenog režima „zamenskog odlučivanja“, po kome se osobi sa mentalnim invaliditetom oduzima mogućnost da 30 https://documents-dds-ny.un.org/doc/UNDOC/GEN/G14/031/20/PDF/G1403120. pdf?OpenElement. Miloš Janković, Lišavanje slobode u ustanovama socijalne zaštite 289 shodno sopstvenoj volji (želji, htenju) donosi odluke koje se nje tiču, nameće joj se neko drugi ko donosi odluke umesto nje, uključujući i suprotno njenoj volji. Takva odluka je zasnovana na nekakvom objektiviziranom „najboljem interesom“, a ne na autentičnoj volji i sklonostima određene osobe. Stav Komiteta je da zamensko odlučivanje treba zameniti „paradigmom podrške odlučivanju“, odnosno „potpomognutom odlučivanju“, te je u tom smislu neophodno izvršiti unapređenje teksta Nacrta. 4.2. Deinstitucionalizacija U odredbi naslovljenoj „Razlozi smeštaja“ navedeno je da se „smeštaj... obezbeđuje korisniku kome se trenutno ne može obezbediti ostanak u porodici, dnevne usluge u zajednici ili usluge podrške za samostalan život u smislu zakona kojim je uređena socijalna zaštita“. Navedena odredba je iznuđena i oslikava realno stanje koje se ogleda u činjenici da u Srbiji ne postoje uslovi da se većini korisnika obezbede adekvatni životni uslovi van institucija. Očigledno je da je na tom planu malo učinjeno. Poražavajuće deluje da se Nacrtom zakona Vladi daje rok od od godinu dana da usvoji „planski dokument koji će se odnositi na dnevne usluge u zajednici ili usluge podrške za samostalan život“. Kašnjenje u sprovođenju deinstitucionalizacije u Srbiji u suprpotnosti je sa stavovima Komiteta za prava osoba sa invaliditetom ali i sa Strategijom unapređenja položaja osoba sa invaliditetom31, starom preko jednu deceniju, kao i prethodnom, takođe starom preko jednu deceniju i aktuelnom Strategijom za zaštitu mentalnog zdravlja u Srbiji za period 2019–2026.32 Pre svega, svi navedeni dokumenti nalažu neophodnost sveobuhvatne deinstitucionalizacije, što znači prestanak daljeg dugoročnog držanja osoba sa mentalnim smetnjama u glomaznim stacionarnim ustanovama, koje treba smanjiti i postepeno gasiti. U tom cilju neophodno je uspostaviti kako lečenje, tako i održiv sistem zbrinjavanja osoba sa mentalnim smetnjama u zajednici, u njima odgovarajućem okruženju koje nije određeno njihovim mentalnim stanjem, uz pružanje svakodobne i svakovrsne, kontinuirane multifunkcionalne podrške, kako za njih, tako i za i njihove porodice. Na žalost, na tom planu je malo učinjeno. Nasuprot tome, Nacrt zakona stvara pravnu osnovu da svi postojeći korisnici s mentalnim smetnjama koji su smešteni u ustanovama socijalne zaštite ostanu u njima, mnogi do kraja života. U prilog tome govori i odredba Nacrta u kojoj se propisuje da će se o vaninstitucionalnoj zaštiti osobe sa mentalnim smetnjama odlučivati tek u postupku njenog smeštaja u ustanovu socijalne zaštite. 31 32 Na str. 12. navedeno je da je potrebno „obezbediti da sistem socijalnih, zdravstvenih i drugih usluga za osobe sa invaliditetom u potpunosti poštuje princip dostupnosti usluga u lokalnoj zajednici, uz potpunu primenu procesa deinstitucionalizacije; na strani 13. naglašava se da treba „promovisati porodično okruženje kao primarno i najbolje za osobu sa invaliditetom uz punu primenu i snažnu podršku deinstitucionalizaciji. Na strani 2. navedeno je da „velike psihijatrijske i socijalne ustanove moraju biti smanjene, a sistem zaštite mentalnog zdravlja reformisan. Uz postojeće resurse potrebni su novi da bi se podržao proces reformi, posebno uspostavljanje daljeg razvoja lečenja i zaštite mentalnog zdravlja u zajednici, što bi omogućilo lečenje u sredini u kojoj osobe sa mentalnim poremećajima žive, uz primenu najmanje restriktivnog pristupa u lečenju. 290 CRIMEN (X) 3/2019 • str. 278–298 4.3. Lišavanje slobode korisnika u ustanovama socijalne zaštite Osnovna dilema postoji u pogledu opravdanosti prisilnog smeštanja, odnosno lišavanja slobode korisnika u ustanovama socijalne zaštite. Sporno je da li karakter ustanova socijalne zaštite, koje postoje da bi svojim korisnicima pružale usluge, dozvoljava da se u njima sprovodi prinudno smeštanje, odnosno lišavanje slobode i njihovo izopštavanje iz društva. Deluje kao neodrživa konstrukcija da lišavanje slobode predstavlja „uslugu” korisniku socijalne zaštite. Sledeći tu logiku, i sputavanje korisnika vezivanjem bi predstavljalo uslugu. Čini se da prisila, ili bilo koji vid ograničavanja ne može predstavljati uslugu. U pokušaju da izbegnemo donošenje pogrešnih zaključka, na ovom mestu je potrebno da napravimo distinkciju između, s jedne strane, lišavanja slobode, i s druge strane, nametanja minimalnog i prihvatljivog obima ograničenja prema korisnicima usluga u ustanovama socijalne zaštite. Naime, nesporno je da pojedini korisnici (npr. lica sa demencijom) moraju da zbog vlastite bezbednosti budu podvrgnuti određenim ograničenjima. Međutim, ukoliko se odnos prema takvim licima ne zasniva na nametanju ograničenja (prevashodno zaključavanjem i drugim oblicima fizičkog ili hemijskoag sputavanja) već na pružanju pažnje i podrške, te ukoliko im je omogućeno slobodno kretanje po ustanovi, komunikacija i pristup raznovrsnim, njima odgovarajućim sadržajima (shodno kućnom redu koji je restriktivan u najmanje mogućoj meri), kao i ako im se omogućuje da (uz obaveznu pratnju) odlaze van institucije do mesta po svom izboru, radi održavanja porodičnih, socijalnih kontakata, kao i korišćenja kulturnih, sportskih ili drugih sadržaja (u razumnom intezitetu i obimu), takvo postupanje nema karakter lišenja slobode. Ukoliko bi napred opisano lice bilo uglavnom zaključano u ograničenom prostoru i ograničeno u socijalnim kontaktima, naročito ako bi bilo podvrgnuto fizičkom ili hemijskom sputavanju, uz izostanak napred navedenog tretmana, takvo postupanje ima karakter lišavanja slobode. 4.4. Lišavanje slobode osoba sa mentalnim smetnjama u ustanovama socijalne zaštite U tekstu Nacrta zakona nije korišćena uobičajena terminologija poput „nedobrovoljni smeštaj”, „prinudni smeštaj” ili „lišavanje slobode”, a „smeštaj bez pristanka” se spominje na nekoliko mesta, i to u nereferentnom kontekstu po ovom pitanju. Ono što navedeni izrazi jasno oslikavaju prikriveno je pod plaštom izraza „postupak smeštaja u slučaju nedostatka volje” i „postupak smeštaja na osnovu odluke suda”. Ilustrativan pokazatelj je odredba naslovljena „Smeštaj korisnika”, u kojoj se u prvom stavu propisuje smeštaj „na osnovu pristanka“ a u drugom stavu, u kome se suštinski govori o smeštaju bez pristnka, odnosno prinudnom smeštaju, ti izrazi se ne navode, već se koristi se opisna terminologija „smeštaj se obezbeđuje i licu koje nije u stanju da iskaže svoj pristanak“. Miloš Janković, Lišavanje slobode u ustanovama socijalne zaštite 291 Kao što je napred ukazano, slično je i sa očigledno smišljenim izostavljanjem izraza „osobe sa mentalnim smetnjama” iz teksta Nacrta zakona. Na primer, iz propisanih uslova za prinudno smeštanje u ustanovu socijalne zaštite nesumnjivo proističe da te uslove kumulativno ispunjavaju jedino osobe sa mentalnim smetnjama, a koristi se izraz „korisnik“. Shodno tome, možemo zaključiti da Nacrt Zakona sadrži odredbe koje se odnose na prisilno smeštanje, odnosno lišavanje slobode osoba sa mentalnim smetnjama u ustanovama socijalne zaštite, iako je to jezički prikriveno. Smeštanje osoba sa mentalnim smetnjama u ustanove socijalne zaštite, naročito ako je ono prinudno, je sporno. Osobama sa mentalnim smetnjama je prevashono potrebno lečenje i u tom pogledu u Srbiji postoji, s jedne strane, zakonski okvir koga čini postojeći Zakon o zaštiti osoba sa mentalnim smetnjama i brojni podzakonski propisi za njegovo sprovođenje, a sa druge strane, i adekvatne psihijatrijske ustanove koje su podobne za lečenje osoba sa mentalnim smetnjama. Smeštanjem osoba sa mentalnim smetnjama u ustanove socijalne zaštite zanemaruje se potreba njihovog specijalizovanog lečenja i života u bliskom socijalnom, kulturnom i drugom njima odgovarajućem okruženju. Oni se izopštavaju iz sredine u kojoj su do tada živeli ili u kojoj žele da žive, a nameće im se život u instituciji po pravilima institucije i uz odsustvo adekvatnog lečenja i brojna ograničenja kojima su izloženi, što sve može doprineti daljem urušavanju njihovog mentalnog zdravlja, 4.5. Postupak lišavanja slobode u ustanovama socijalne zaštite U odredbi naslovljenoj „Postupak u slučaju nedostatka volje“, propisani su uslovi za pokretanje postupka pred sudom radi prinudnog smeštaja. Možemo ih grupisati u tri celine: 1) da lice nije sposobno da razume značaj i posledice izjave koju daje, odnosno nije sposobno da izrazi svoju volju; 2) da lice svojim postupcima ugrožava sopstvenu bezbednost, život, zdravlje, egzistenciju, prava i interese i bezbednost, život, prava i interese drugih lica; 3) da mu se ne mogu obezbediti ili nije u njegovom interesu pružanje usluga socijalne zaštite dnevne usluge u zajednici, usluge podrške za samostalan život, porodični smeštaj i druge usluge kojima se korisniku omogućava da živi u porodici ili samostalno. Prvo pitanje koje se nameće je šta se dešava sa licem o čijem „smeštaju” se odlučuje počev od momenta kada centar za socijalni rad proceni da su ispunjeni napred propisani uslovi i obrati se sudu, pa sve do momenta donošenja pravosnažne, odnosno izvršne odluke od strane suda. Osim toga, imajući u vidu da je propisano da su uslovi kumulativni, odnosno da svi moraju biti ispunjeni, te da je očigledno da je za brižljivu multidisciplinarnu stručnu procenu ispunjenosti svakog pojedinčnog uslova potrebno značajno vreme, postavlja se i dodatno pitanje šta se dešava sa tim licem u prethodnom periodu, odnosno počev od momenta kada Centar za socijalni rad dođe u neposredan kontakt sa tim licem, pa sve do momenta sačinjavanja konačne procene i formalnog obraćanja sudu. U oba navedena slučaja je nejasno da li će lice o čijem „smeštaju” se odlučuje u opisano vreme biti privremeno prinudno smešten u ustanovu socijalne zaštite, a 292 CRIMEN (X) 3/2019 • str. 278–298 ako hoće, po kom osnovu. Ako neće, nepoznanica je da li to znači da će lice za koje postoji sumnja da može ugroziti sebe ili drugog biti pušteno ili će biti odvedeno u psihijatrijsku ustanovu. Takođe, kada je u pitanju kumulativnost uslova postavlja se i pitanje npr. šta se dešava sa licem koje je „sposobno da razume značaj i posledice izjave koju daje“, odnosno ako nije ispunjen prvi uslov za prinudni smeštaj, a ispunjena su ostala dva. Shodno jasnoj odredbi, takvo lice ne može biti prinudno smešteno. U takvom slučaju, u kome je procenjeno da to lice npr. ugrožava bezbednost drugih lica, a ne želi da bude smešten u ustanovu socijalne zaštite, uopšte nije propisano kako treba da postupiti centar za socijalni rad. I u ovom slučaju je nepoznanica da li će to lice, za koje postoji sumnja da može ugroziti sebe ili drugog, biti pušteno ili će biti odvedeno u psihijatrijsku ustanovu. Dalje, nije određeno ko procenjuje, odnosno ko daje stručno mišljenje da li je lice sposobno da „razume značaj i posledice izjave koju daje“ i „izrazi svoju volju“, kao i da li lice „svojim postupcima“ ugrožava sebe ili drugog. Nije precizirano da li je to psihijatar i da li on mora imati status veštaka. Osim toga nedostaju i kriterijumi koji su odlučni za donošenje takve procene, naročito u pogledu eventualnog ugrožavanja bezbednosti, života, zdravlja, egzistencije, prava i interesa. Ta odredba Nacrta zakona je sporna i u pogledu toga što propisuje dužnost centra za socijalni rad da u slučaju ispunjenosti svih uslova za prinudni smeštaj ujedno „kod suda pokrene postupak za lišenje poslovne sposobnosti“. Navedenim se ozakonjuje već ustanovljena praksa, odnosno trend da sva lica koja su prinudno smeštena u ustanovama socijalne zaštite ujedno budu i lišena poslovne sposobnosti. Takva situacija može biti i predmet zloupotreba i mnoge korisnike dovesti u stanje tzv. civilne smrti. Posebno zabrinjava da u postupku procene pred centrom za socijalni rad korisnicima nisu dostupni zaštitni mehanizmi koji shodno važećim standardima moraju biti uspostavljeni. Između ostalog, nije propisano da osoba koja je predmet procene mora da bude neposredno saslušana, da mora da bude pregledana od strane nezavisnog psihijatra, da mora da joj se obezbedi pravna zaštita putem izabranog advokata ili kroz pružanje besplatne pravne pomoći, te da u postupku mora da se omogući učešće članova porodice i kao drugih bliskih lica koje ta osoba odabere. Odredbom je propisano da će se u ustanove socijalne zaštite prinudno smestiti lica koja kumulativno ispunjavaju sve postavljene uslove. Iz propisanih uslova nesumnjivo proističe da je reč o osobama sa mentalnim smetnjama. Kao što je napred rečeno, takvim licima je pre svega neophodno lečenje i u tom pogledu postoji adekvatan zakonski okvir koga čini postojeći Zakon o zaštiti osoba sa mentalnim smetnjama i brojni podzakonski propisi za njegovo sprovođenje, kao i adekvatne ustanove, specijalizovane psihijatrijske bolnice i odeljenja opštih bolnica, koje su podobne za lečenje osoba sa mentalnim smetnjama. U istoj oderdbi je propisano postupanje u „u slučaju smeštaja kod pružaoca usluge .... koje je tu dovedeno bez svog pristanka... radi donošenja odluke suda“. Postavlja se pitanje po kom osnovu bi se neko lice, bez njegovog pristanka, dovodilo kod „pružaoca usluge“. Nisu određeni uslovi da bi se to preduzelo, ko je ovlašćen Miloš Janković, Lišavanje slobode u ustanovama socijalne zaštite 293 da to čini i po kom postupku. Zakon o zaštiti prava osoba sa mentalnim smetnjama propisuje takvu situaciju, ali predviđa za dovođenje u zdravstvenu ustanovu, a ne u ustanovu socijalne zaštite. Dalje, u toj odredbi se uređuje situacija kada je lice smešteno u ustanovi socijalne zaštite na osnovu „svog pristanka... ili pristanka zakonskog zastupnika, pa je taj pristanak povučen pisanom izjavom“. Nejasno da li će se u takvoj situaciji tom licu omogućiti da napusti ustanovu, ako neće po kom osnovu će on biti prinudno zadržan u ustanovi. Takođe, pitanje je kako će se postupiti prema licu koje je smešteno u ustanovi socijalne zaštite na osnovu „svog pristanka“, ali odluči da napusti ustanovu, ne želeći da piše bilo kakvu izjavu. Nejasno je da li će se to lice prinudno zadržati u ustanovi, a ako hoće, po kom osnovu. Na kraju, šta će biti sa detetom koje je smešteno u ustanovi socijalne zaštite na osnovu „pristanka zakonskog zastupnika“, u situaciji kada pristanak nije „povučen pisanom izjavom“ zakonskog zastupnika, a dete, koje može imati npr. 17 godina, hoće da napusti ustanovu. I ovde se postavlja pitanje da li će to lice biti prinudno zadržano u ustanovi, a ako hoće, po kom osnovu. Poslednji stav te odredbe, kojim je propisno da sud „u okviru postupka iz stava 1. ovog člana sud pribavlja...“, tu se našao očiglednom greškom. Naime „postupak iz stava 1.” završava se kada centar za socijalni rad „kod suda pokrene postupak“. Jasno je da sud ne može da „pribavlja“ bilo šta pre no što je pred sudom pokrenut postupak. 4.6. Sputavanje U odredbi Nacrta zakona naslovljenoj „Odlučivanje o sputavanju“ propisano je da „odluku o sputavanju korisnika donosi psihijatar, uz konsultacije sa... zaposlenima kod pružaoca usluge“. Potpuno je nejasno koji je značaj takvih konsultacija i u kojoj meri su one relevantne za odluku psihijatra, nejasno je da li je on u tom pogledu vezan za mišljenje lica koje konsultuje. U istoj odredbi je propisano da „ako razlozi za sputavanje postoje kada psihijatar nije dostupan, a okolnosti zahtevaju da odluka o sputavanju bude doneta hitno, odluku o sputavanju može doneti stručni radnik, stručni saradnik ili zdravstveni saradnik angažovan kod pružaoca usluge, koji je u tom slučaju dužan da psihijatra odmah obavesti o sputavanju i zatim postupi u skladu sa njegovim uputstvima“. Pre svega, navedena odredba ima nedostatak logičke prirode. Naime „kada psihijatar nije dostupan“, osoblje pružaoca usluge neće moći ni da ga obavesti o sprovedenom sputavanju niti će moći od njega da pribavi uputstva po kojima treba da postupi. Dalje, situacija „kada psihijatar nije dostupan“ nije u skladu sa odredbom koja propisuje „sputavanje je dozvoljeno samo u ustanovama koje imaju obezbeđene sve potrebne uslove za njegovu primenu“. Za pretpostaviti je da je uslov za primenu sputavanja svakodobna dostupnost psihijatra, te ukoliko određenoj ustanovi psihijatar nije dostupan, onda ta ustanova ne ispunjava uslove za primenu sputavanja. 294 CRIMEN (X) 3/2019 • str. 278–298 Razumljivo bi bilo da je Nacrtom zakona propisano da osoblje ustanove može sputati agitirano lice vezivanjem u situaciji kada psihijatar nije prisutan na licu mesta, kada je to urgentno i neophodno, međutim psihijatar mora uvek biti dostupan i u mogućnosti da u najkraćem roku dođe do sputanog lica, te da proceni da li je neophodno njegovo dalje sputavanje, kao i da lice periodično nadzire sve vreme dok je sputano. Sputavanje korisnika od strane osoblja i njihovo dalje postupanje prema sputanom licu u skladu sa uputstvima psihijatra dobijenim preko sredstava telekomunikacije ili ako je ono unapred dato je nedopustivo. 4.7. Izdvajanje U odredbi naslovljenoj „Značenje izraza“ i „Vrste sputavanja“ kao jedan od načina sputavanja predviđeno je „izdvajanje u posebnu prostoriju“. To predstavlja izolaciju, izuzetno restriktivnu meru koja podrazumeva potpuno odsustvo socijalnog kontakta, što može imati pogubne posledice na mentalno zdravlje lica prema kome je mera primenjena, a naročito ako je osoba sa mentalnim smetnjama. Specijalni izvestilac UN za torturu je u svom izveštaju iz 2011. godine ukazao da izolacija osoba sa mentalnim smetnjama, u bilo kom trajanju, predstavlja surovo, nečovečno ili ponižavajuće postupanje i da države treba da ukinu meru usamljenja prema osobama sa mentalnim smetnjama33, a Potkomitet UN za prevenciju torture je u svom izveštaju o poseti Paragvaju iz 2010. godine uputio preporuku da uopšte ne treba vršiti izolaciju osoba sa mentalnim smetnjama.34. Shodno napred navedenom, odredbe iz Nacrta zakona koje se odnose na „izdvajanje“ korisnika treba brisati. 4.8. Sputavanje farmakoterapijom U odredbi naslovljenoj „Značenje izraza“ i „Vrste sputavanja“ kao jedan od načina sputavanja predviđeno je „hemijsko sputavanje“ koje se obezbeđuje „sprečavanjem ili ograničavanjem kretanja lica davanjem farmakoterapije“. Takav pristup je potpuno u skladu sa praksom upotrebe lekova u ustanovama socijalne zaštite. Korisnicima se lekovi kao redovna terapija namenski daju radi sprečavanja ili ograničavanja njihovog kretanja, na koji način se uspostavlja njihova statičnost koja pogoduje ostvarivanju kontrole od strane uglavnom nedovoljo prisutnog osoblja. Međutim smisao sputavanja farmakoterapijom nije i ne sme biti kontiunirano „sprečavanje ili ograničavanje kretanja“ korisnika, već umirivanje agitiranog lica kako bi se otklonila opasnost da ugrozi sebe ili drugog. Uočeno je da prihijatri često ostavljaju na ocenu dežurnom osoblju na ocenu da li određenom korisniku da daju određene lekove. Farmakoterapiju, i to isključivo radi umirenja agitiranog korisnika treba da određuje isključivo psihijatar, u svakom 33 34 Privremeni izveštaj Generalnom sekretaru /2011/, [A/66/268 (2011)], t. 68, 78 i 86. Izveštaj o poseti Paragvaju /2010/, [CAT/OP/PRY/1 (2010)], t. 185. Miloš Janković, Lišavanje slobode u ustanovama socijalne zaštite 295 konkretnom slučaju, u zavisnosti od konkretnih okolnosti. Propisivanje terapije unapred i ostavljanje na ocenu (na način: „po potrebi“) drugom, medicinskom pa i nemedicinskom osoblju nije prihvatljivo. 4.9. Ograničenje slobode kretanja i drugih prava korisnika U odredbi naslovljenoj „Sloboda kretanja“ propisano je da „korisnik ima pravo na slobodu kretanja, osim u slučajevima u kojima se ovo pravo ograničava pravilima pružaoca usluge...“. Ovde je potrebno ponovo ukazati da se prava ne mogu ograničavati pravilima, time ni „pravilima pružaoca usluge“. Član 20. Ustava Republike Srbije propisuje da se „ljudska prava zajemčena Ustavom mogu zakonom ograničiti“, i to „samo ako ograničenje dopušta Ustav“, a član član 27. shodno tome propisuje da je „lišenje slobode dopušteno samo iz razloga i u postupku koji su predviđeni zakonom”. U narednoj odredbi, naslovljenoj „Ograničenje prava“, između ostalog je predviđeno ograničavanje prava „usled zdravstvenog stanja korisnika“. Zdravstveno stanje ili stanje invaliditeta ne smeju biti razlog za bilo kakvo ograničavanje prava. U odredbi naslovljenoj „Ostvarivanje ljudskih prava“ navedeno je da „korisnik usluge smeštaja ima pravo na ostvarivanje građanskih, političkih, ekonomskih, socijalnih, kulturnih i drugih prava, u skladu sa Ustavom i zakonom“. Navedeno je puka proklamacija jer je nesporno da lica koja su prinudno smeštena u ustanovama socijalne zaštite u najvećoj meri nisu u mogućnosti da ostvaruju sva nabrojana prava poput svih ostalih lica. Umesto generalizovanja, bolje bi bilo da je Nacrtom zakona zajamčeno i propisan način ostvarivanja konkretnih prava koja se korisnicima uobičajeno uskraćuju. To bi na primer moglo da bude aktivno biračko pravo, imajući u vidu da će iz člana 52. Ustava Srbije u najskorijem roku morati da bude brisana poslovna sposobnost kao uslov za ostvarivanje izbornog prava. 4.10. Pritužba i prigovor U odredbi naslovljenoj „Pritužba“ utvrđeno je da pravo na pritužbu imaju „korisnik, član njegove uže porodice ili zakonski zastupnik koji smatra da mu je pružalac usluge onemogućio ostvarenje nekog prava propisanog zakonom, koji nije zadovoljan kvalitetom pružanja usluge, postupkom ili ponašanjem pružaoca usluge, ili drugog korisnika, kao i nekim drugim pitanjem u vezi sa pružanjem usluge“. U odredbi naslovljenoj „Prigovor“ propisano je da podnosioci pritužbe koji nisu zadovoljni ishodom „mogu podneti prigovor organu nadležnom za vršenje inspekcijskog nadzora“. Mogućnost ulaganja pravnog leka na tu odluku, odnosno sudska kontrola tih akata nije propisana. Očigledno je da je Nacrtom zakona propisan niži nivo pravne zaštite za lica lišena slobode u ustanovama socijalne za štite, od onog koji je za osuđena lica lišena slobode propisan Zakonom o izvršenju krivičnih sankcija. Zaista neprimereno deluje da sistem socijalne zaštite korisnicima nudi niži nivo pravne zaštite nego u sistemu izvršenja krivičnih sankcija, u kome je, osim sudske kontrole upravnih akata donetih po pritužbama lica lišenih slobode, ustanovljena i posebna kontrola specijalizovanog „sudije izvršenja“. 296 CRIMEN (X) 3/2019 • str. 278–298 4.11. Uloga policije U odredbi kojom je propisana „Pomoć policije“, navodi se da će policija biti pozvana da „pronađe i u ustanovu vrati“ korisnika koji je „bez znanja i odobrenja“ napustio ustanovu, a koji je u njoj „smešten bez pristanka“, ali i korisnika „koji nije u stanju da razume o čemu se radi“. To što je obaveza policije da „pronađe i u ustanovu vrati“ ne samo prinudno smeštene korisnike, već i one koji su u njoj zvanično dobrovoljno smešteni, pokazatelj je postojeće prakse koja se želi legalizovati. Naime, pojedini korisnici, koji su registrovani kao dobrovoljno smešteni (na osnovu postojanje njihovih potpisa ili potpisa nihovih staralaca na aktima o prijemu), faktički su smešteni i držani u ustanovi mimo njihove volje. Jasno je da ne postoji objašnjenje zašto bi policija vraćala u ustanovu korisnika koji u njoj nije prinudno smešten. Korisnik koji je dobrovoljno smešten u ustanovu može je napustiti kada god to želi. Shodno tome, nejasno je zašto je Nacrtom zakona policiji dato ovlašćenje i dužnost da „pronađe i u ustanovu vrati“ lice koje je u njoj bilo smešteno svojom voljom. 4.12. Shodna primena zakona U posebnoj odredbi Nacrta zakona propisan je „postupak smeštaja na osnovu odluke suda”, a u odredbi o „shodnoj primeni propisa“ utvrđena je shodna primena „zakona kojim je uređena zaštita lica sa mentalnim smetnjama, a na pitanja postupka koja nisu posebno uređena tim zakonom primenjuju se odredbe zakona kojima se uređuje vanparnični postupak“. Navedenim je učinjena potpuna konfuzija. Pre svega, nejasno je koji je domašaj propisivanja shodne primene „Zakona kojim je uređena zaštita lica sa mentalnim smetnjama“. Navedenim su dodatno učinjene nejasnim odredbe kojim je Nacrtom zakona propisan „postupak smeštaja na osnovu odluke suda”. Nepotpuno i neprecizno propisivanje postupka, uz pozivanje na shodnu primenu druga dva zakona, mogao bi dovesti do nejednake primene prava, pa i zloupotrebe prava na štetu korisnika koji bi se prisilno smeštali u ustanove socijalne zaštite. Ograničenje prava i sloboda mora biti zakonom uređeno na sveobuhvatan, precizan i jasan način, kako sa materijalnog, tako i sa procesnog aspekta. U tom smislu, nužno je da se utvrde konkretni uslovi koji moraju biti ispunjeni da bi neko bio lišen slobode, da se propiše procedura koja mora biti sprovedena, da se utvrdi ko je nadležan za donošenje privremenih odluka pre no što sud započne postupak, rok u kome sud mora biti obavešten, da se u postupku omogući učešće onoga o čijoj slobodi i pravima se odlučuje bez obzira na njegove mentalne kapacitete, da mu se o trošku države omogući pravna pomoć, da nakon neposrednog suočavanja sudije sa tim licem, na osnovu stručnog mišljenja nezavisnog psihijatra konačnu odluku o lišavanju slobode donese sud, da postoji mogućnost žalbe na tu odluku, kao i obaveza da se u shodnoj proceduri vrši njeno periodično preispitivanje u razumnim rokovima. Miloš Janković, Lišavanje slobode u ustanovama socijalne zaštite 297 ZAKLJUČAK U ustanovama socijalne zaštite domskog tipa je smešten veliki broj korisnika koji su uglavnom zaključani ili na drugi način onemogućeni da svojevoljno napuste te ustanove, odnosno faktički su lišeni slobode. Takvo postupanje je u suprotnosti sa odredbama Ustava Republike Srbije i najvažnijih instrumenata međunarodnog prava. Ministarstvo nadležno za socijalna pitanja je pokušalo da reši navedeni problem donošenjem zakona koji će predstavljati pravni osnov za smeštanje korisnika u ustanove socijalne zaštite bez njihove saglasnosti. Tokom 2019. godine sačinjene su dve verzije Nacrta zakona, prva je naslovljena „Zakon o zaštiti lica sa mentalnim smetnjama u socijalnoj zaštiti”, a druga „Zakon o zaštiti prava korisnika usluga smeštaja u socijalnoj zaštiti“. Ignorisana je osnovna dilema u pogledu opravdanosti prisilnog smeštanja, odnosno lišavanja slobode korisnika u ustanovama socijalne zaštite. Sporno je da li karakter ustanova socijalne zaštite, koje postoje da bi svojim korisnicima pružale usluge, dozvoljava da se u njima sprovodi prinudno smeštanje, odnosno lišavanje slobode i njihovo izopštavanje iz društva. Pošavši od toga da je nezakonito lišavanje slobode nedozvoljeno i očigledno pod teretom zadatka da se zakonom održi postojeće stanje i da se očuvaju postojeće ustanove socijalne zaštite, pisci Nacrta zakona su pokušali da ponuđenim rešenjima legalizuju dosadašnju praksu i da normiranjem postupanja prema korisnicima prinudno smeštenim u tim ustanovama stvore privid da će na taj način biti povećan nivo njihove zaštite. Analizom teksta Nacrta zakona može se zaključiti da u tome nisu uspeli. Naime očigledno je da ponuđene verzije Nacrta zakona sadrže brojne nedostatke i da su u suprotnosti sa važećim standardima. LITERATURA I OSTALI IZVORI Janković M. /2015/: Slobode i prava lica sa mentalnim smetnjama – nedostaci Zakona o zaštiti lica sa mentalnim smetnjama, Crimen (VI) 1. Janković M. /2018/: Položaj osoba sa mentalnim smetnjama lišenih slobode, Sociologija Vol. LX, n° 4. Janjić B., Ćirić Milovanović D: /2017/: Ovde i zidovi imaju uši, Beograd, Inicijativa za prava osoba sa mentalnim invaliditetom MDRI-S. Jović V. /2015/: Opšta psihopatologija sa konceptualnom istorijom, K. Mitrovica, Filozofski fakultet Univerziteta u Prištini. Kecmanović D. /2008/: Psihijatrija u kritičkom ogledalu, Beograd, Službeni glasnik. Mole N., Auer F. i Braithwaite B. /2014/: Međunarodni i uporedni standardi – in: Prava osoba sa mentalnim invaliditetom (S. Gajin, ed.), Beograd, Centar za unapređenje pravnih studija. Godišnji izveštaj Zaštitnika građana i NPM za 2017. godinu. Izveštaj o poseti Paragvaju /2010/, [CAT/OP/PRY/1 (2010)]. 298 CRIMEN (X) 3/2019 • str. 278–298 Izveštaj Zaštitnika građana o poseti domu Veternik, br. 71 –26/ 13. Izveštaj NPM za 2017. godinu. Privremeni izveštaj Generalnom sekretaru /2011/, [A/66/268 (2011)]. Strategija za zaštitu mentalnog zdravlja u Srbiji za period 2019–2026. Svetska zdravstvena organizacija /2010/: Deseta revizija Međunarodne statističke klasifikacije bolesti i srodnih zdravstvenih problema. Zakon o ratifikaciji Međunarodnog pakta o građanskim i političkim pravima („Službeni list SFRJ“, br. 7/1971–73). Zakon o ratifikaciji Evropske konvencije za zaštitu ljudskih prava i osnovnih sloboda („Službeni list SCG – Međunarodni ugovori“, br. 9/2003 i 5/2005). Zakon o ratifikaciji Konvencije o pravima osoba sa invaliditetom („Sl. glasnik RS – Međunarodni ugovori“, br. 42/2009). Zakon o ratifikaciji Opcionog protokola uz Konvenciju UN protiv torture i drugih surovih, neljudskih ili ponižavajućih kazni ili postupaka („Službeni list SCG – Međunarodni ugovori“, br. 16/2005 i 2/2006). Zaštitnik građana, Mišljenje 12–3077/13. Zaštitnik građana, Preporuka Opštoj bolnici u Šapcu 61 – 1885 / 14. Miloš Janković DEPRIVATION OF LIBERTY IN SOCIAL WELFARE INSTITUTIONS SUMMARY A large number of beneficiaries who are accommodated in social welfare homes are persons with mental disabilities. Most of them are locked up and prevented from leaving those institutions by their own will. They are virtually deprived of their liberty, with the explanation that it is in their “best interest”. However, there is no legal ground in the Serbian legal system for the deprivation of liberty of beneficiaries of social welfare institutions. Such treatment is contrary to Article 27 of the Serbian Constitution according to which “deprivation of liberty is allowed only for the reasons and in the procedure provided for by law”. It also contravenes Article 5 of the European Convention on Human Rights, according to which “no one shall be deprived of his liberty save... in accordance with a procedure prescribed by law”. After years of ignoring the Ombudsman’s recommendation to stop deprivation of liberty without valid legal ground, the Ministry responsible for social affairs in 2019 tried to regulate the said conduct. However, the offered versions of the Draft Law contain numerous shortcomings and contradict current standards. This paper is an attempt to argue for the unsustainability of the solutions offered. Key words: deprivation of liberty, social care institutions, involuntary accomodation, mental disability, torture, ill-treatment, restraining 

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For more information visit www.intechopen.com Chapter 1 Planning Primary Total Knee Arthroplasties João Bosco Sales Nogueira, Leonardo do Carmo Araújo and João BoscoHeráclio Sales Nogueira, Marcelo José CortezdoBezerra Leonardo Heráclio Carmo Araújo and Marcelo José Cortez Bezerra Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.72775 Abstract Preoperative planning is routinely recommended prior to total knee arthroplasty (TKA). We introduce a methodology for planning TKA based on mechanical alignment. A methodology for planning total knee arthroplasty was discussed among experienced knee surgeons. A rational methodology for planning TKA was stablished and it was setted to an application for mobile devices. It has proved to be useful and revealed accuracy compared to the manual form of preoperative planning. It was able to reduce planning time by more than a half and it was still reliable in measuring the anatomical-mechanical femoral angle (MAFÂ). This chapter introduces a TKA planning method based on mechanical alignment and GAP balancing principles. Kinematic alignment and strategies for soft-tissue balancing in special situations are cited as well. Keywords: knee surgery, arthroplasty, knee injuries surgery, mobile applications operative time 1. History and introduction The very first concept of improving knee function was introduced by Verneuil in 1860. He proposed an interposition of soft tissues in order to reconstruct articular surface. Unfortunately, it has led to disappointing results. In the same year, Ferguson resected the entire surface of the knee, which culminated with a better range of motion but lacked stability. In 1958, MacIntosh described a hemiarthroplasty using an acrylic tibial plateau later upgraded by McKeever for a prosthesis made of metal, showing better results. Guston developed a polycentric prosthesis wich arculates metal to a polyethylene base fixed to the bone by acrylic cement, but the actual concepts used in total knee arthroplasty (TKA) have been established by Freeman in 1973: minimal bone resection; minimal chance of loosening; © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, Commons Attribution (http://creativecommons.org/licenses/by/3.0), and reproduction in any License medium, provided the original work is properly cited. which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 Primary Total Knee Arthroplasty minimal debris production; not leaving dead spaces; avoiding long intramedullary stem and intramedullary cement; a standard insertion procedure; minimal range of motion (5–90°); resisting rotation; and resisting excessive movements in any direction. During the early 1970s, a range of prostheses such as unicondylar, bicondylar, and hinged were used with respect to the patient’s preoperative condition and deformity. Since then, a lot of different kinds of implants have been developed following the tendency of maximizing flexion, minimizing wear, and better accommodation of gender and racial anatomic variation. Nowadays, a resurrection of old strategies such as uncemented fixation and partial knee replacement has been noted and minimally invasive approaches are growing respecting the patient’s desire of shortening postoperative recovery [1, 2]. This chapter introduces a TKA preoperative planning method based on mechanical alignment and the modified GAP balancing principles. Kinematic alignment (KA) principle, anterolateral approach, and strategies for soft tissue balancing in special situations are cited as well. 2. Biomechanics and templating The most important surgical technique that affects patient satisfaction and functional outcomes is the correct positioning and alignment of the components. Despite the introduction of computer assisted surgery, patient-specific designs, and kinematic knee alignment, the concept of optimal restoration of alignment still has added controversy on the best approach of planning TKA [3–5]. Most surgeons still agree, and it is traditionally accepted that the alignment of the lower limb should be within 0° ± 3° of the mechanical axis after surgery (measured by the angle formed by the center of the femoral head, the center of the knee, and the center of ankle) [6] (Figure 1). By positioning the femoral and tibial components perpendicular to its mechanical axis, there is a balanced distribution of mediolateral forces, not overloading the bone implant interface as well as the bone itself which could lead to loosening of the implant. Furthermore, malalignment causes increased polyethylene wear, leading to osteolysis. In order to plan a TKA, a preoperative image exam that evidences all reference points is necessary to estimate the anatomical and mechanical axis of the femur and tibia. Since short-leg X-rays do not expose the whole lower limb references, they are not suitable for planning TKA when it is used alone. Long-leg radiograph (LLR) is traditionally used to estimate alignment of the lower limb. It is acquired on weight-bearing films, including the hip, knee, and ankle, with the patella facing forwards. The X-ray beam should be parallel to the articular surface [7]. Although the LLR acquisition is quite standardized, has long track records and it is fairly available, there are concerns about its use, since flexion contractures and rotational malposition could distort the interpretation of the native anatomy [8]. Besides the fact that LLR is quite standardized, has a long track record, and is fairly available, there have been reported concerns about the accuracy when there are flexion contractures and when the position of image acquisition is not neutral, since rotation of the limb could distort the interpretation of native anatomy. Planning Primary Total Knee Arthroplasties http://dx.doi.org/10.5772/intechopen.72775 Figure 1. Alignment angle. Measured by the center of the femoral head to center of the knee and center of the ankle. It should be within 0° ± 3°. 2.1. Manual templating For this instance, we propose a step-by-step methodology for planning TKA based on mechanical alignment, using LLR. We suggest the use of a parchment paper as it avoids scratching the LLR. First, the mechanical axis of the femur (MAF) is defined by connecting a line from the center of the femoral head to the femoral intercondylar notch. Then, the anatomical axis of the femur (AAF) is estimated by connecting a line from the center of the intramedullary femoral canal to the femoral intercondylar notch. The angle formed by the mechanical and anatomical axis of the femur (MAFÂ) is measured, using a protractor. This part of planning has particular importance for those who intend to use intramedullary distal femoral cut guides. Since the femoral stem follows the path of the AAF, the surgeon would know how to accomplish a 3 4 Primary Total Knee Arthroplasty perpendicular cut to the MAF, by positioning the distal femoral cut guide in a valgus angle to the AAF, referenced by MAFÂ. After knowing the inclination of the distal femoral cut, the surgeon should evaluate the level of bone resection, anticipating the amount of bone resection. Usually, in a primary TKA, the level of bone resection corresponds to the femoral component thickness (e.g., 9 mm). This step simulates the surgical moment when the surgeon places the distal femoral cut guide onto the distal femoral bone. Using a protractor, the surgeon draws a line perpendicular to the MAF above the first point of the distal femoral bone contact (e.g., 9 mm), from distal to proximal. By doing it, the surgeon could simulate the level and inclination of the distal femoral cut. One should notice that the LLR film does not correspond to real size, being reduced to fit on the film frame. Typically, the amount of reduction in size is reported on the printed film and should be used to make a conversion between film size and actual size. The same rationality is applied to the tibial bone. The mechanical and anatomical axes are coincident on the tibia, so the mechanical axis of the tibia (MAT) can be defined as a line drawn from the center of the tibial spines to the center of the talus. Mechanical and anatomical axes are usually coincident on the tibia. The mechanical axis of the tibia (MAT) can be defined as a line drawn from the center of the tibial spines to the center of the talus. After defining MAT, tibial cut is planned perpendicular to the MAT. Usually, surgeons plan to cut 8–10 mm below the unworn side of the tibial plateau, but it can be adjusted to best fit the components and balance the knee. So, the surgeon places a line from proximal to distal, at the level desired below the unworn side of the plateau. Still, conversion between LLR size and real size should be adjusted using a mathematical calculation. The whole process of manual templating technique is illustrated in Figure 2. The more parallel the bone cut planes, the less ligament release is necessary. The less parallel the bone cut planes, the more ligament release is necessary. One must note that there is a limit for releasing ligaments. When a great amount of releasing is necessary, the surgeon should consider a more constrained implant, since it could get into a situation called “over resection looseness,” when the ligament function capacity is exceeded. The size of the components can be also estimated during the planning of a TKA. To do it so, the surgeon must have the specific prosthesis templates and should order a short-leg X-ray in an actual size (1:1). 2.2. Digital planning using ATJ® mobile phone application Despite the fact that manual planning is an affordable method, it lacks portability. The surgeon must have manual tools such as a pencil; an eraser; a protractor; and rulers, big enough to measure the whole LLR film. Besides, the surgeon needs to make mathematical conversions and must comprehend a rational methodology of planning on TKA. Considering the current computational resources used as tools to support medical practices, the paradigm known as mHealth, which consists of the use of mobile computing resources in health, stands out. When applied to favor teaching and learning processes, mobile technologies provide numerous benefits, such as increased resources for student learning, access to Planning Primary Total Knee Arthroplasties http://dx.doi.org/10.5772/intechopen.72775 Figure 2. Manual templating. First, the anatomical and mechanical axis of the femur and tibia are defined. MAFÂ is measured by using a protractor. A perpendicular cut is measured from MAF and MAT. To correctly place the bone cut lines, the surgeon must mathematically convert printed LLR film size to real size. To do it, the surgeon needs manual tools such as pencil, eraser, rulers, protector, and goniometer. textbooks anywhere, anytime, and the provision of resources for the development of innovative teaching methods [9]. By facilitating real-world integration, mobile learning aided by mobile computing (Mobile Learning or m-learning) has created opportunities for development of new teaching strategies in different areas [10]. An interdisciplinary team, involving health and computer science areas, developed an application (ATJ®) that is capable of conducting the surgeon though a step-by-step process (same described 5 6 Primary Total Knee Arthroplasty Figure 3. Fields of the medical register. They are available offline and online, in a cloud host. Planning Primary Total Knee Arthroplasties http://dx.doi.org/10.5772/intechopen.72775 Figure 4. After choosing between the left or right knee, the surgeon must inform the application in which size scale is adopted on LLR. By doing so, the surgeon does not need to make mathematical calculations, since the application is able to convert LLR size to real size. for manual planning) adapted for digital planning. Besides, explanations about rationality of the methodology and surgical tips on each step of planning were incorporated to the application. It is able to recognize the type of deformity (valgus or varus) and automatically measure MAFÂ. It also suggests soft tissue releases, according to the deformity [11]. 7 8 Primary Total Knee Arthroplasty Figure 5. The LLR should be uploaded from the device’s camera roll or the device’s camera can be used to acquire the LLR image (Figure Planning). Before getting into planning, the surgeon is presented with a video that briefly describes the method used in the application. Fields of registration such as name, email, telephone, age, gender, medical record number, and date are available. The data are stored on the device and in a cloud host. Anytime, wherever the surgeon is, it is possible to access it from any computer if desired (Figure 3). Planning starts by choosing the intended knee to be operated (left or right). After choosing the side, the application inquiries on size scale used on LLR, which is usually informed on LLR margins. This is an important step since the application needs to know the relation between the LLR film size and real size, avoiding the surgeon making mathematical conversions (Figure 4). The LLR can be acquired by two modes: uploading an image from device’s camera roll or using the device’s camera to photograph the LLR on a negatoscope (Figure 5). The application Planning Primary Total Knee Arthroplasties http://dx.doi.org/10.5772/intechopen.72775 Figure 6. Step-by-step planning process. After defining AAF, MAF, MAFÂ, and MAT, the surgeon should simulate the level and inclination of distal femoral and proximal tibial bone cuts. Application ATJ® is capable of automatically recognizing the knee-type deformity. It also suggests a sequence for ligament releasing. then takes the surgeon through the whole process of planning by inquiring the surgeon to point references (center of the femoral head, center of the medullary femoral canal, center of the intercondylar notch, center of the tibial spines, and center of the talus). After setting points, the application automatically defines AAF, MAF, MAFÂ, and MAT. It also positions a 9 10 Primary Total Knee Arthroplasty moveable bone cutting line, perpendicular to MAF and MAT. At the end of the methodology process, it automatically shows the type of deformity, the MAFÂ, and soft tissue releasing sequence (Figure 6). Then, scientific references used to build the application are presented. The application ATJ® has proved to be useful in the context of planning TKA. It has revealed accuracy when measuring the MAFÂ when compared to the manual form of planning. It was also capable of reducing the planning time by more than a half [11, 12]. 3. Surgery 3.1. Surgical approaches A straight anterior skin incision allows exposure for medial and lateral structures and it’s the standard skin incision for TKA. Arthrotomy may be done from the medial or lateral side. Anteromedial arthrotomy is the most used approach on TKA and provides excellent exposure of the knee joint (Figure 7). After eversion or patella lateral subluxation, the knee is flexed. One must be aware of avulsing off the patellar tendon from the tibial tubercle, if there is too much tension. Because the most important component of blood supply runs medially, anteromedial approach could affect patellar circulation, and some authors have advocated subvastus and midvastus approaches (Figure 8). They are less invasive and can be used specially in moderate knee deformity. For fixed valgus deformities, the lateral parapatellar approach may be considered. A mild lateral skin incision is made and extended over the lateral border of the tibial tubercle. In the original description, a thin segment of the tubercle is osteotomized with the attached patellar tendon. A medial periosteal hinge is maintained along with the infrapatellar fat pad, which is used for later closer of the lateral retinacula. Some surgeons suggest not to osteotomize the tibial tubercle, turning it into a less invasive approach. Extended approaches have been described and are very useful in stiff knees and great deformities. Coonse and Adams described a quadriceps turndown. The quadriceps are split down the middle, in an inverted “V” fashion, at about 1 cm above the patella, so the patella could be turned down, allowing the exposure of the joint. Quadriceps snip was described by Insall by an oblique incision at the proximal apex of the quadriceps tendon, at about a 45° angle, directly in line with the fibers of the vastus lateralis. It relaxes the extensor mechanism and protects the tibial tubercle. Tibial tubercle osteotomy enhances exposure and it could be very useful in stiff knees. Tibial crest should be osteotomized 8–10 cm below the tibial tubercle, using the oscillating saw and osteotomes. The shape of osteotomy is trapezoidal, 5-m long, 2-cm wide, and 1.5-cm wide distally. The entire extensor mechanism is then elevated proximally. Limited approaches are described and could be useful, especially when planning unicompartmental arthroplasties. Limited approaches are part of a traditional extensile approach, and the surgeon tends to use it as he becomes familiar with the surgical technique. Planning Primary Total Knee Arthroplasties http://dx.doi.org/10.5772/intechopen.72775 Figure 7. Anteromedial approach to the knee. After a medial skin incision, a medial parapatellar arthrotomy is made. It is suitable for most TKA and allows great exposure of the knee joint. Figure 8. Subvastus approach does not sacrifice the extensor mechanism (blue line) and it is useful specially for minimal invasive approaches. In midvastus approach (red line), the oblique medial vastus is split sharply in line with its fibers, at the level of the superior pole of the patella. 3.2. Surgical theories 3.2.1. Anatomical, mechanical alignment, and gap balance technique Since the beginning of the implants and instruments, two concepts that guide surgical techniques were established: gap balance and measured resection technique. Universally spread, gap balance surged for cruciate substituting prostheses. Measured resection technique was favored for those who defended cruciate retention implants. For gap balance technique, either the femur or tibia may be osteotomized first. The main goal is to equalize flexion and extension gaps, most of the time transforming a trapezoidal gap into a rectangular gap (Figure 9). For those who favor to start cutting proximal tibial (at about 8–10 mm below the less worn tibial plateau surface), it should be perpendicular to the MAT, and when posterior cruciate ligament (PCL) is sacrificed, the flexion gap opens up a few millimeters more. That situation can implicate in a slightly upper distal femur cut, which can elevate the joint line and bring the patella in a lower position. Distal femoral cut is often parallel to the tibial cut, following the transepicondylar axis. Rotational alignment of the femur is tuned by ligament release or femoral rotation. In a varus knee, when the surgeon opts for 11 12 Primary Total Knee Arthroplasty Figure 9. Before ligament releasing, there is usually a trapezoidal gap. not releasing medial ligaments he usually needs to add external rotation to the femoral cuts. Some references help to establish the appropriate rotation: transepicondylar axis, posterior femoral condyles, Whitside’s line (trochlear groove axis), tibial shaft axis, and ligament tension. In some situations, even a small degree of internal rotation could be applied. Before implant is settled, the gap must be rectangular and symmetrical (Figure 10). The measured resection technique aims not to move the joint line position. That situation, in theory, preserves knee’s anatomy, sparing the PCL, when possible. Some advocate that preserving the PCL has advantages as it is an important varus/valgus stabilizer of the knee; it can absorb stress; and it can control the movement of rolling back of the femur onto the tibia Figure 10. Rectangular gap. Before the implant is set, there must be a rectangular gap. Planning Primary Total Knee Arthroplasties http://dx.doi.org/10.5772/intechopen.72775 during flexion. When the ligament is left too tight it could increase posterior stress, causing a movement described by Insall as “open like a book” and “lift-off” of the tibial tray. When the PCL is insufficient, it could cause a paradoxal movement of rolling forward in flexion, limiting flexion by posterior impingement. The tibial cut is made preserving its mild varus and femur cut is made preserving its natural valgus. Equalization of the gaps is not strictly necessary. During the evolution of the implants some concepts were mixed and the surgeon conquered freedom to make bone cuts a little bit off the outliers. Even for situations when the PCL is preserved, conformed tibial surfaces could be used. Sometimes, the surgeon can choose not to cut the bone perpendicular to the mechanical axis, sparing extensive ligament releases. 3.2.2. Kinematic alignment The refinement of surgical techniques, implant designs and an individualized tendency of surgical treatment have contributed to resurrection of the discussion about restoration of the native’s knee anatomy and preservation of the articular line. Some authors have noticed that neutral mechanical alignment does not restore biomechanics in a significant part of the population [13–15]. Kinematic Alignment (KA) aims to restore constitutional alignment, ligament tension, and the joint’s line level and orientation [16, 17]. Hungerford, Kenna, and Krakow defended a slight varus alignment for the tibia in relation to the MAT. They are considered precursors on KA [18]. According to Howell et al., there are three kinematic knee axis: the primary femoral axis, wich is a transverse axis of femur around which the tibia flexes and extends; the secondary femoral axis, wiich is a traverse axis in the femur around which the patella flexes and extends; and the longitudinal tibial axis, around which the tibia internally or externally rotates on the femur [19] (Figure 11). Each axis is parallel or perpendicular to the natural joint line between the femur and tibia throughout the motion arc. The preoperative plan of a kinematic TKA can be done by using MRI exams in order to estimate chondral and bone erosion [20–22]. These parameters are used to compensate implant’s position that should be parallel to the primary and secondary kinematic femoral axis. A patient-specific implant or a conventional implant can be used. The KA principle is a promising alternative for the execution of TKA. In the centers where it was adopted, the results in the short and medium term were favorable [22], with the premise of restoration of the biomechanics of the knee, which may point to a new paradigm. Nevertheless, studies on durability and long-term function are needed before universal adoption of this new methodology. Its applicability in patients with a higher degree of bone erosion should also be evaluated cautiously because of the increased risk of malalignment, which may be caused by the difficulty in identifying the references that guide the positioning of the bone cutting guides. 13 14 Primary Total Knee Arthroplasty Figure 11. Kinematics axis of the knee (from Howell’s studies with authorization). 4. Management of deformities 4.1. Varus deformity During the arthritic course, there are expected modifications in native alignment and ligament function. Loss of cartilage can create an asymmetrical compartment balance, leading to contracture of soft tissues on the concave side and contralateral loosening on the convex side. As the deformity progresses, these modifications tend to establish into fixed deformities. In varus knees, contraction of medial side involves medial collateral ligament and the whole medial periosteal sleeve, including hamstrings, posteromedial capsule, and PCL. As the deformity progresses, the lateral compartment becomes insufficient, causing abnormal lateral opening and instability. Instability of the arthritic knee may be viewed as symmetrical or asymmetrical. Symmetrical instability is seen during early arthritis, when there is erosion of cartilage or bone without associated adaptive soft tissue changes. During physical examination, the deformity can be corrected under active reciprocal stress on physical examination. This kind of instability is easily corrected during surgery without needing extensive ligament releasing. As the deformity progresses it tends to turn into an asymmetrical instability, which is not corrected by active reciprocal stress during physical examination. This kind of fixed deformity occurs when cartilage and bone loss lead to adaptive ligamentous changes. In order to correct this kind of deformity, ligamentous release is mandatory, turning a trapezoidal gap into a rectangular gap. In asymmetrical instabilities, the bone cuts alone are not sufficient to accomplish articular balance. Instead of only releasing ligaments from the contracted concave side someone could advocate to advance ligament complexes on the convex side, especially when the opposing ligaments are stretched to the point of being incompetent. These authors do not favor ligament advances or reconstructions on the convex side since the functional outcomes have not been acceptable in most series. In such cases, the authors advise considering a more constrained knee design (Figure 12). Planning Primary Total Knee Arthroplasties http://dx.doi.org/10.5772/intechopen.72775 Figure 12. In advanced deformities, when there is great medial bone loss, the surgeon must consider using metal augmentation and more constrained implants. 4.2. Valgus deformity In the valgus knee, the lateral structures contract, while the medial soft tissues stretch. Differing from varus knees, where the bone erosion occurs more in the tibial bone, in valgus knees most of the bone deformity comes from the femoral side (Figure 13). As the deformity progresses it tends to involve the tibial bone and cartilage as well. Lateral soft tissue structures, including the lateral collateral ligament (LCL), iliotibial band (ITB), and the lateral capsule, contract, while the medial soft tissues stretch. When this Figure 13. Most part of the bone wear occurs on lateral femoral condyle, although it could involve tibial bone as well. 15 16 Primary Total Knee Arthroplasty imbalance becomes permanent it may result in medial thrust during gait. In both varus and valgus deformities, it could be associated with flexion contractures (Figure 14). Krackow has classified valgus deformities into three stages: type I involves lateral femoral bone loss, lateral soft tissue contracture, and intact medial soft tissues; type II adds medial lengthened soft tissues; and type III represents alteration of the proximal tibial joint line, that usually happens as a result of a high tibial osteotomy. As the ITB gets contracted, external rotation is often observed. Insall and colleagues traditionally recommended releasing lateral capsule, LCL, arcuate ligament, popliteus tendon, lateral femoral periosteum, distal ITB, and the adjacent lateral intermuscular septum from their bone attachments. Some degree of lateral laxity after an extensive lateral release was typically well tolerated. Although extensive release generally corrects the deformity, posterolateral flexion instability may still occur postoperatively. These authors do not favor extensive releasing from bone attachments. Instead, the authors recommend progressively intra-articular liberations ahead of the popliteus tendon, using the pie-crusting technique. 4.3. Flexion contracture As the degenerative disease progresses, it involves posterior capsule, PCL, and musculotendinous at the posterior aspect of the knee. Bone erosion of posterior femoral condyles and osteophytes may contribute to flexion contracture on arthritis. Despite the fact that some authors understand that postoperative residual flexion contractures are well tolerated, these authors do not favor residual flexion contractures, since residual deformities tend to worsen with time. Figure 14. Valgus deformity. Contraction of the lateral compartment causes bone erosion and an imbalance of natural kinematics. Planning Primary Total Knee Arthroplasties http://dx.doi.org/10.5772/intechopen.72775 After bone cuts, posterior capsule can be released. For those who agree with principles of KA, the preservation of PCL is desirable. That could hamper flexion contracture correction but changing the slope orientation could in part correct that kind of deformity. Small contractures can be reduced by removal of posterior osteophytes, but posterior capsulotomy is necessary for moderate and severe flexion contractures. These authors advise the resection of PCL when the mechanical alignment theory is chosen. 4.4. Extension contracture Stiff knees are a challenging situation even for the most experienced knee surgeons. Typically, the techniques described for difficult exposures are necessary. Sometimes, everting the patella is not possible, leading the surgeon only to subluxate it laterally. The soft tissue releases applied for varus and valgus techniques are used and extensive soft tissue release is usually necessary. 4.5. Genu recurvatum Recurvatum is usually mild and it’s treated with under-resection of the distal femoral and proximal tibia. However, in patients with neuromuscular diseases, such as poliomyelitis and Ehler-Danlos, even under-resection and use of thicker components are insufficient to correct recurvatum. For these extreme conditions, the authors recommend the use of constrained prosthesis or hinged implants. 5. Management of bone defects As osteoarthritis advances, bone defects can distort the natural anatomy of the knee and cause to difficult alignment and implant set. Figure 15. Peripheral medial bone defect on medial tibial plateau. One must avoid over-resection of the medial tibial plateau as it could fracture proximal fibula. Cancellous bone is insufficient to support the tibial implant. 17 18 Primary Total Knee Arthroplasty Contained defects occur from a cyst or a cavity and are treated by filling off the defect or bone cut. One must remember that there is a limit for moving the bone cut level. Augmentation with cement, bone, or metal wedges or blocks should be considered in these cases. Peripheral defects typically occur in varus knees in the posteromedial plateau and in valgus knees in the distal lateral femur. These kinds of defects are also managed with cement, bone graft, and metal augments. When they occur in tibia, the translation of the tibial tray away from the location of the defect could be sufficient. If not deeper than 10 mm, the defect can be eliminated by resecting the tibia at a lower level until, at a maximum of, 20 mm, but these authors recommend using metal augmentation and intramedullary stems to protect the implant from interface shear forces (Figure 15). 6. Choice of implant There are theoretical concerns when choosing the right implant. For those who choose anatomical and kinematic alignment, the implant tends to reproduce the knee’s anatomy and the surgeon should preserve at the most ligaments and the natural inclination of the native’s articular line. In such cases, the PCL should be preserved whenever it’s possible, since it is a varus/valgus stabilizer, and it can absorb stress. Anterior cruciate ligament (ACL) plays a role with PCL during knee flexion and extension. As the knee flexes, the femur slides back in tibia (rollback) until a point where the ACL is completely strengthened. As the knee extends, the femur slides forward until a point that the PCL resists this movement. For arthrosis of one compartment, when ALC and PCL are intact, a meniscal-bearing design could be used (e.g., Oxford® unicompartmental prosthesis). As arthrosis progresses, ACL becomes insufficient. When PCL is not sacrificed, the movement of rollback occurs and theoretically, the tibial baseplate should be flat. When PCL is sacrificed, the tibial baseplate should be concave, containing forward and backward motion. However, some of the newer implants now allow PCL sacrifice or retention, regardless the shape of tibial baseplate. Mobile-bearing designs have increased the sagittal plane conformity which helps to control anteroposterior translation. The increased coronal plane conformity typically presented in mobile-bearing TKA also increases the contact area and lessens contact stresses. These advantages tend to reduce the rate of polyethylene wear. Polyethylene is self-aligned with the femoral component. It reduces the cross-shear stresses and facilitates central patellar tracking. In a fixed-bearing TKA, if the tibial component is left in internal rotation, it moves tibial tuberosity laterally, enhancing the risk of patellar subluxation. Besides, mobile-bearing designs also contribute to diminish the incidence of lateral releasing. Clinically, the fixed-bearing and mobile-bearing TKA systems have performed similarly in outcome studies. These authors favor the use of mobile-bearing designs, especially for younger and higher-demand patients with longer life expectancies. Planning Primary Total Knee Arthroplasties http://dx.doi.org/10.5772/intechopen.72775 7. Emerging technologies Patient-specific instruments could be made from preoperative imaging (MRI or CT scans of specific sequences). It could allow the production of manufactured specific cutting guides. These guides are made in respect of the individual anatomy, including osteophytes and bone defects in the correct orientation. It would direct the bone resection before the preplanned knee, avoiding using standard intraoperative cutting guides. Specific guides should require fewer trays of kit, leading to greater operative efficiency, and are expected to reduce operative time and produce more accurate bone cuts. At this point, there are a lot of studies that suggest the use of specific guides with good functional outcomes. Nevertheless, cost-effectiveness still needs to be proven. Robotic surgery combines navigation with a robot that performs the bony resection, controlled by the surgeon. A preoperative CT scan is used to template the knee. It has a theoretical advantage of not deviating from the defined cutting plane or axes of resection. Despite the appeal that it would reproduce better mechanical axis, additional studies are necessary to justify its use outside the experimental environment before it gets universally used. 8. Conclusion The restoration of adequate mechanical axes is critical for implant survival. Preoperative planning anticipates surgical difficulties and gives a chance for creating resolutive strategies. The ATJ® application for mobile phones has proven useful and comes to optimize the surgical planning in TKAs. As it establishes a rational step-by-step process, based on literature, it directs the user to a possible reliable form of surgical planning. Author details João Bosco Sales Nogueira1*, Leonardo Heráclio do Carmo Araújo2 and Marcelo José Cortez Bezerra3 *Address all correspondence to: bosco.nogueira@centrodojoelho.com.br 1 Medical School of Federal University of Ceará, Fortaleza, Brazil 2 Hospital Geral de Fortaleza/Santa Casa da Misericórdia de Fortaleza, Fortaleza, Brazil 3 Universidade de Fortaleza/Santa Casa de Fortaleza, Fortaleza, Brazil 19 20 Primary Total Knee Arthroplasty References [1] Scott WN. Insall and Scott Surgery of the Knee. New York: Elsevier; 2012 [2] Ranawat AS, Ranawat CS. The history of total knee arthroplasty. Knee Joint [Internet]. 2012;26:699-707. Available from: http://link.springer.com/10.1007/978-2-287-99353-4_63 [3] Donaldson J, Joyner J, Tudor F. Current controversies of alignment. In: Scott WN, editor. Insall and Scott Surgery of the Knee. New York: Elsevier; 2015. pp. 489-494 [4] Yoon J-R, Yang J-H. Satisfactory short-term results of navigation-assisted gap-balancing total knee arthroplasty using ultracongruent insert. Journal of Arthroplasty [Internet]. 2017:1-6. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0883540317308513 [5] Singh AK, Nedopil AJ, Howell SM, Hull ML. Does alignment of the limb and tibial width determine relative narrowing between compartments when planning mechanically aligned TKA? Archives of Orthopaedic Trauma Surgery [Internet]. 2017. Available from: http://link.springer.com/10.1007/s00402-017-2824-6 [6] Deakin AH, Sarungi M. A comparison of variable angle versus fixed angle distal femoral resection in primary total knee arthroplasty. Journal of Arthroplasty [Internet]. 2014;29(6):1133-1137. Available from: http://linkinghub.elsevier.com/retrieve/pii/ S0883540313008541 [7] Babazadeh S, Dowsey MM, Bingham RJ, Ek ET, Stoney JD, Choong PFM. The long leg radiograph is a reliable method of assessing alignment when compared to computerassisted navigation and computer tomography. Knee. [Internet]. 2013;20(4):242-249. DOI: http://dx.doi.org/10.1016/j.knee.2012.07.009 [8] Wu W, Guo WS, Cheng LM, Liu ZH, Zhang QD, Zhang NF. Individual difference of coronal bowing of femur and its influence on the lower limbs alignment after the total knee arthroplasty. Zhonghua Yi Xue Za Zhi. 2017 Apr 4;97(13):1006-1010. DOI: 10.3760/cma. j.issn.0376-2491.2017.13.009. [Article in Chinese; Abstract available in Chinese from the publisher] [9] Marçal E, Andrade R, Rios R. Aprendizagem utilizando Dispositivos Móveis com Sistemas de Realidade Virtual. RENOTE Revista Novas Tecnologias na Educacao [Internet]. 2005;3:1-11. Available from: http://seer.ufrgs.br/renote/article/view/13824 [10] Huang T et al. Total knee arthroplasty in patients with Ranawat type-II valgus arthritic knee with a marked coronal femoral bowing deformity: Comparison between computer-assisted surgery and intra-articular resection. Journal of Orthopaedic Surgery and Research. 2016;11:1-9 [11] Nogueira JBS, de Carvalho ACGS, de Barros Filho EM, do Araújo LHC, Bezerra MJC. Planejamento de artroplastia total do joelho através de aplicativo para dispositivos móveis: Relato de caso. Rev Bras Ortop. 2017:1-7 Planning Primary Total Knee Arthroplasties http://dx.doi.org/10.5772/intechopen.72775 [12] Nogueira JBS, de Carvalho ACGS, de Barros Filho EM, do Araújo LHC, Bezerra MJC, Demange MK. Avaliação de utilidade e acurácia de aplicativo móvel no planejamento de artroplastias totais do joelho. Revista Brasileira Ortopedia [Internet]. 2017:1-9. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0102361616302090 [13] Bellemans J, Colyn W, Vandenneucker H, Victor J. The chitranjan ranawat award. Clinical Orthopaedics and Related Research. 2012;470(1):45-53 [14] Nedopil AJ, Singh AK, Howell SM, Hull ML. Does calipered kinematically aligned TKA restore native left to right symmetry of the lower limb and improve function? Journal of Arthroplasty [Internet]. 2017. DOI: https://doi.org/10.1016/j.arth.2017.09.039 [15] McNair PJ, Boocock MG, Dominick ND, Kelly RJ, Farrington BJ, Young SW. A comparison of walking gait following mechanical and kinematic alignment in total knee joint replacement. Journal of Arthroplasty [Internet]. 2017. Available from: http://linkinghub. elsevier.com/retrieve/pii/S0883540317308331%0A; http://www.ncbi.nlm.nih.gov/pubmed/ 29054726 [16] Howell SM, Howell SJ, Hull ML. Assessment of the radii of the medial and lateral femoral condyles in varus and valgus knees with osteoarthritis. Journal of Bone and Joint SurgeryAmerican Volume [Internet]. 2010;92(1):98-104. Available from: http://content.wkhealth. com/linkback/openurl?sid=WKPTLP:landingpage&an=00004623-201001000-00013 [17] Howell SM, Chen J, Hull ML. Variability of the location of the tibial tubercle affects the rotational alignment of the tibial component in kinematically aligned total knee arthroplasty. Knee Surgery, Sport Traumatology, Arthroscopy. 2013;21(10):2288-2295 [18] Toksvig-Larsen S, Ryd L, Stentström A, Dansgård F, Jonsson K, Robertson O, et al. The porous-coated anatomic total knee experience. Journal of Arthroplasty [Internet]. October 8, 2017;11(1):11-17. DOI: http://dx.doi.org/10.1016/S0883-5403(96)80156-2 [19] Howell SM, Papadopoulos S, Kuznik KT, Hull ML. Accurate alignment and high function after kinematically aligned TKA performed with generic instruments. Knee Surgery, Sport Traumatology Arthroscopy. 2013;21(10):2271-2280 [20] Howell SM, Kuznik K, Ghaly R, Hull ML. Does varus alignment adversely affect implant survival and function six years after kinematically aligned total knee arthroplasty? International Orthopaedics. 2015;39(11):2117-2124 [21] Nam D, Lin KM, Howell SM, Hull ML. Femoral bone and cartilage wear is predictable at 0o and 90o in the osteoarthritic knee treated with total knee arthroplasty. Knee Surgery, Sport Traumatology Arthroscopy. 2014;22(12):2975-2981 [22] Howell SM, Howell SJ, Kuznik KT, Cohen J, Hull ML. Does a kinematically aligned total knee arthroplasty restore function without failure regardless of alignment category? Knee. Clinical Orthopaedics and Related Research. 2013;471(3):1000-1007 21 

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ЭКОНОМИЧЕСКИЕ И ЮРИДИЧЕСКИЕ НАУКИ. Юридические науки УДК 342.5(438) № 3(65) DOI 10.52928/2070-1632-2023-65-3-75-78 ДЗЯРЖАЎНА-ПРАВАВАЯ ХАРАКТАРЫСТЫКА ЛЮБЛІНСКАЙ УНІІ 1569 ГОДА А.В. ВАЛЕЎКА (Полацкі дзяржаўны ўніверсітэт імя Еўфрасінні Полацкай) ORCID: https://orcid.org/0000-0003-4258-8095 У артыкуле разгледжаны асноўныя палажэнні акта Люблінскай уніі 1569 года; дадзена прававая адзнака дзяржаўнай мадэлі Рэчы Паспалітай, згодна са зместам акта Уніі;вызначаны асаблівасці дзяржаўнага ладу Рэчы Паспалітай; праведзены аналіз навуковых поглядаў на сутнасць і гісторыка-прававое значэнне Люблінскай уніі; згодна з палажэннямі акта Уніі зроблена выснова аб палітыка-прававым стане Вялікага Княства Літоўскага ў складзе Рэчы Паспалітай. Ключавыя словы: Люблінская унія, Рэч Паспалітая, Вялікае Княства Літоўскае, сойм. Уводзіны Дзяржаўны лад Рэчы Паспалітай з’яўляецца аб’ектам даследавання вучоных на працягу доўгага часу. Люблінская унія як дакумент канстытуцыйнага характару мела выключнае значэнне ў працэсе замацавання асноў дзяржаўнага ладу Рэчы Паспалітай. Менавіта падрабязнае даследаванне зместу акта уніі дазваляе выявіць асаблівасці Рэчы Паспалітай як дзяржаўнага ўтварэння, а таксама знайсці прычыны тых супярэчнасцей, якія суправаджалі дзяржаву на працягу яе існавання. Асноўная частка Рэч Паспалітая ўзнікла ў выніку пагаднення паміж Вялікім Княствам Літоўскім і Польскім каралеўствам, вядомаго як Люблінская унія 1 ліпеня 1569 года (далей – Унія). Унія мела канстытуцыйны характар, вызначаючы асновы палітычнага ладу новай дзяржавы. Гістарычнае значэнне дадзенага дакумента ў розных крыніцах ацэнена па-рознаму. Некаторыя аўтары разлядалі Унію з негатыўных пазіцый. Напрыклад, І. Лаппо адзначаў, што нягледзячы на замацаваны ў 3 артыкуле акта Уніі прынцып раўнапраўнага аб’яднання Польшчы і Вялікага Княства Літоўскага, на практыцы гэты прынцып так і не быў рэалізаваны. Сама Унія ў’яўляла сабой фармальны дакумент, які кожны з бакоў імкнуўся выкарыстаць ва ўласных інтарэсах [1, с. 8]. Я. Юхо ў сваіх працах адзначаў, што Унія была навязаная Вялікаму Княству Літоўскаму амаль гвалтоўна і вяла да павелічэння ўплыву Польшчы і паланізацыі мясцовага насельніцтва [2, с. 136]. Нераўнапраўны характар Уніі Я. Юхо падцвярджае фактам адабрання Польшчай некаторых тэрыторый Вялікага Княства Літоўскага з мэтай схілення літоўскага боку да заключэння пагаднення. А. Грыцкевіч адзначаў, што да заключэння Уніі імкнулася шляхта Вялікага Княства Літоўскага, якая на сойме 1562 г. накіравала вялікаму князю просьбу аб заключэнні дзяржаўнай уніі паміж Польшчай і Вялікім Княствам Літоўскім [3, с. 412]. Такім чынам Унія разглядаецца як вынік узаемадзеяння шэрагу аб’ектыўных фактараў. На наш погляд, Унія стала новым этапам у развіцці палітычных і ваенных адносін паміж Польшчай і Вялікім Княствам Літоўскім і з’яўлялася вынікам палітычных імкненняў некалікіх бакоў, аднак гэтая спроба не была дасканалай і не магла пераадолець супярэчнасці, якія існавалі паміж дзвюма дзяржавамі. Так ці інакш, асновай канстытуцыйна-прававога рэгулявання і ўпарадкавання дзяржаўна-прававых адносін Рэчы Паспалітай стала Унія – дакумент палітычнага і прававога характару, які вызначаў асновы канстытуцыйнага ладу новай дзяржавы, асноўныя прынцыпы ў сістэме ўзаемаадносін асоба – грамадства – дзяржава. Уступнай часткай акта адзначалася важнасць захавання папярэдніх пагадненняў паміж Вялікім Княствам Літоўскім і Польшчай. У першую чаргу на ўвазе маюцца Крэўская і Гарадзельская уніі, якія былі пачатковымі эпатамі ў развіцці саюзных узаемаадносін паміж дзвюма краінамі. Такім чынам, Люблінская унія павінна была ўмацаваць ужо існуючыя адносіны Вялікага Княства Літоўскага і Польшчы. Таксама важна звярнуць увагу на тое, што акт уніі не ігнараваў прывілеі і граматы, якія былі выдадзены манархамі абедзвюх дзяржаў да моманту заключэння пагаднення. Змест першага артыкула акта Уніі вызначае асноўныя характарыстыкі новай дзяржавы – Рэчы Паспалітай – як адзінай і непадзельнай, утворанай на аснове двух народаў і дзвюх дзяржаваў [4]. Другі артыкул замацоўваў фактычна існуючы на той момант стан манарха: адна асоба для Вялікага Княства Літоўскага і Польшчы. Разам з тым, быў прадугледжаны парадак абрання караля Рэчы Паспалітай агульным галасаваннем прадстаўнікоў Польшчы і ВКЛ. Такім чынам Рэч Паспалітая павінна была стаць выбарнай манархіяй. Зыходзячы са зместу наступнага артыкула, можна зрабіць выснову, што нашчадкі караля таксама мелі права прымаць удзел у выбарах караля ў якасці аднаго з кандыдатаў. Пры іх неабранні гарантавалася матэрыяльная падтрымка з боку дзяржавы. Новаабраны кароль павінен быў падцвердзіць прывілеі і правы саслоў’яў Рэчы Паспалітай. У будучым гэта палажэнне было рэлізавана ў форме актаў «Pacta Conventa», якія абавязкова прымаліся пры абранні караля. Згодна артыкулу 6 у Рэчы Паспалітай складвалася адзіная прадстаўнічая сістэма, асновай якой павінен быў стаць агульнадзяржаўны сойм. Асабістыя соймы для Вялікага Княства Літоўскага і Польшчы не прадугледжваліся. Гэтае палажэнне таксама было падцверджана ў артыкуле 15. Прадугледжвалася ўзаемападтрымка паміж станамі Вялікага Княства Літоўскага і Польшчы ў палітычных, ваенных, эканамічных і іншых справах. У Вялікім Княстве Літоўскім, згодна з артыкулам 14, захоўваліся былы адміністрацыйны апарат, асобнае ад Польшчы заканадаўства і судовая арганізацыя, назва дзяржавы «Вялікае княства Літоўскае» і дзяржаўная 75 2023 ВЕСТНИК ПОЛОЦКОГО ГОСУДАРСТВЕННОГО УНИВЕРСИТЕТА. Серия D пячатка, а таксама войска. Была ўстаноўлена забарона на аднабаковае ўхваленне пагадненняў, саюзаў з іншымі дзяржавамі. Гэта тычылася і ваенных пытанняў. Такім чынам знешняя палітыка Рэчы Паспалітай таксама павінна была стаць адзінай і ўсе рашэнні прымаліся сумесна літоўскім і польскім бакамі. Артыкулы 17 і 18 аб’яўлялі ненарушнасць і недатыкалінасць уласнасці як на тэрыторыі ВКЛ, так і на тэрыторыі Польшчы. Артыкулам 25 была ўстаноўлена адказнасць за парушэнне патрабаванняў уніі. Згодна з ім той, хто парушыў адно з палажэнняў акта уніі, з’ўляўся ворагам для дзяржавы і ўсіх станаў. Акт Уніі замацоўваў сістэму органаў дзяржаўнай улады Рэчы Паспалітай. Вышэйшым органам улады згодна з артыкулам 15 акта Уніі станавіўся агульны сойм, які мог збірацца на тэрыторыі Польшчы. Асобных соймаў як для Кароны, так і для Княства не прадугледжвалася. Соймы падраздяляліся на звычайныя, што склікаліся каралём, і надзвычайныя. Надзвычайныя соймы мог склікаць кіраўнік каталіцкай царквы ў Рэчы Паспалітай – прымас (арцыбіскуп Гнезненскі) у выпадку смерці караля або яго адмаўлення ад трона [4]. Для выбрання новага караля склікаліся тры соймы: канвакацыйны, элекцыйны і каранацыйны. На канвакацыйным сойме вызначалі час і месца выбараў караля, выпрацоўвалі ўмовы дагавора з кандыдатамі на прастол. На элекцыйным сойме праводзілі выбары і заключалі пагадненне. Урачыстая каранацыя і прысяга караля ажыццяўляліся на каранацыйным сойме. Такім чынам, кажучы «сойм», мы маем на ўвазе не проста заканадаўчы орган, а шырокае паняцце, якое аб’ядноўвае некалькі палітычных інстытутаў. У склад паўнамоцтваў сойма ўваходзілі не толькі пытанні заканадаўчай дзейнасці, але таксама і абранне караля Рэчы Паспалітай, кантроль над дзейнасцю манарха. Таксама сойм меў некаторыя кадравыя паўнамоцтвы выяўленыя ў прызначэнні на пасады вышэйшых службовых асоб. Кампетэнцыя сойма распаўсюджвалася і на судовую дзейнасць. На перыяд працы сойма з ліку дэпутатаў фарміраваўся соймавы суд, які разгладаў найбольш важныя справы. Такім чынам сойм аб’ядноўваў у сабе заканадаўчыя, выканаўчыя і судовыя функцыі. Па сваёй структуры Сойм быў двухпалатным органам і складаўся з Сената Рэчы Паспалітай і Пасольскай Ізбы. У склад сената ўваходзілі прыдворныя саноўнікі, каталіцкія біскупы, вышэйшыя земскія службовыя асобы – ваяводы і кашталяны. Чальцы сената прызначаліся пажыццёва і фактычна неслі адказнасць толькі перад Рэччу Паспалітай. Першапачаткова ў склад Сената ўваходзілі [5, с. 287]: – усе сенатары сойма Польскага каралеўства: 2 архібіскупа, 7 біскупаў, 15 ваявод, 17 «старэйшых» і 49 «малодшых» кашталянаў, 5 вышэйшых службовых асоб; – ад Вялікага Княства Літоўскага: 2 біскупа (віленскі і жамойцкі), 9 ваявод (віленскі, трокскі, смаленскі, полацкі, наваградскі, віцебскі, берасцейскі, мсціслаўскі і менскі), стараста жамойцкі, 10 кашталянаў (9 ваяводстваў і 10-ты жамойцкі) і 5 вышэйшых службовых асоб (маршалак земскі, канцлер, падканцлер, падскарбі земскі і маршалак дворны); – ад Каралеўскай Прусіі, якая раней мела ўласныя парламенцкія інстытуты, у Сойм увайшлі 2 біскупа, 3 ваяводы і 3 кашталяна; – ад тэрыторый, інкарпараваных Каралеўствам Польскім ад Вялікага княства Літоўскага, у Сойм увайшлі 2 біскупа (луцкі і кіеўскі), 4 ваяводы (кіеўскі, валынскі, брацлаўскі і падляшскі) і 4 кашталяна. Пасля колькасць сенатараў змянялася ў межах ад 140 да 147 [5, с. 288]. Зыходзячы з вышэйпрыведзеных даных, можна зрабіць выснову, што Сенат быў створаны на аснове аналагічнага структурнага падраздзялення Сойма Польскага каралеўства. Вялікае Княства Літоўскае, хоць і ўваходзіла ў Рэч Паспалітую роўным у палітычным стаўленні з Польшчай, аднак мела значна меншае прадстаўніцтва ў вышэйшым органе дзяржаўнай улады. Першапачаткова ў склад Пасольскай Ізбы ўваходзіла 170 прадстаўнікоў шляхты, з якіх 48 прадстаўлялі Вялікае Княства Літоўскае. Пасля колькасць амбасадараў павялічвалася, галоўным чынам, дзякуючы адміністрацыйна-тэрытарыяльным рэформам, што праводзіліся ў Вялікім Княстве Літоўскам і Польскім каралеўстве [6, с. 405]. Як бачна, прадстаўніцтва Вялікага Княства Літоўскага тут таксама было недастаткова вялікім. Перавага польскіх прадстаўнікоў у заканадаўчым органе Рэчы Паспалітай, безумоўна, уплывала на прыняцце дзяржаўна значных рашэнняў у інтарэсах польскага боку. У прыватнасці, праблемы функцыянавання агульнага сойма праявіліся ў самым пачатку існавання Рэчы Паспалітай. У. Падалінскі адзначае, што ўжо 2 ліпеня 1569 г. на Люблінскім сойме ўзнікла спрэчка паміж пасламі Сандамірскага, Калішскага і Трокскага ваяводстваў адносна парадку іх засядання ў палаце Пасольскай ізбы, што паралізавала працу сойма і патрабавала непасрэднага ўдзелу вялікага кароннага маршалка, а пазней – і самаго караля Жыгімонта Аўгуста [8, с. 22]. У выніку быў выдадзены адпаведны каралеўскі ліст, тэкст якога, нажаль, не захаваўся. Тым не менш, іерархія земскіх паслоў была вызначана ў прывілеі ад 11 жніўня 1569 г., а таксама ў дадатковым універсале, ухваленым у той жа дзень. Далейшыя канфлікты паміж польскім і літоўскім бакамі патрабавалі прыняцця дадатковых актаў, якія рэгламентавалі асобныя пытанні дзейнасці сойма. Шэраг норм Уніі рэгламентаваў прававы статус караля Рэчы Паспалітай. Манарх быў агульны і згодна з артыкулам 2 павінен быў абірацца роўнымі галасамі польскіх і літоўскіх феадалаў [4]. Тытул вялікага князя ўваходзіў у агульны тытул манарха. Згодна з артыкуламі 6 і 15 манарх меў права склікаць надзвычайныя ці пазачарговыя соймы па пытаннях, якія патрабавалі неадкладнага вырашэння. Пазней прававы статус караля Рэчы Паспалітай больш падрабязна рэгламентаваўся актам, які атрымаў назву «Генрыкавы Артыкулы» (далей – «Артыкулы») – назва ад імя першага элекцыйнага караля Генрыха Валуа. Трэба адзначыць, што ў Рэчы Паспалітай склалася традыцыя, што паміж шляхтай і кожным новым каралём перад яго элекцыяй заключалася спецыяльная дамова – так званая pacta conventa, якая вызначала канкрэтныя абавязкі новаабранага манарха і была 76 ЭКОНОМИЧЕСКИЕ И ЮРИДИЧЕСКИЕ НАУКИ. Юридические науки № 3(65) своеасаблівым індывідуальным кантрактам. Разам з першай pacta conventa былі складзены і «Артыкулы», у якіх выкладаліся падставовыя прынцыпы шляхецкай дзяржаўнасці. Яны абавязалі караля і вялікага князя прызнаваць вольную элекцыю караля, захоўваць рэлігійны мір, не вырашаць пытанняў вайны і міру без сената і сойма, не праводзіць выбараў наступніка пры сваім жыцці (viventerege), склікаць звычайны сойм кожныя два гады, а таксама прадугледжвалася магчымасць правядзення надзвычайных з’ездаў шляхты. Калі шляхта лічыла, што іх правы парушаюцца, то магла адмовіцца падпарадкоўвацца каралю. «Артыкулы» насілі нязменны характар і кожны абраны кароль абавязаны быў іх пацвердзіць [7, с. 525]. У «Артыкулах» абвяшчаліся свабоднае абранне караля, абавязак склікаць сойм раз у два гады тэрмінам на шэсць тыдняў, не склікаць агульнае апалчэнне (паспалітае рушанне) без згоды сойма [7, с. 525]. Нягледзячы на патрабаванні Уніі аб адзінстве новаўтворанай дзяржавы, фактычна кожная частка канфедэрацыі мела свой асобны адміністрацыйны і судовы апарат, сваю прававую сістэму. Гэты стан быў замацованы ў Статуце Вялікага Княства Літоўскага 1588 года. Аб гэтым сведчыць змест артыкула 1 першага раздзела «Усе абывацелі Вялікага Княства Літоўскага гэтым адным правам пісаным і ад нас дадзеным павінны быць судзімыя» [9, с. 14], а таксама артыкул 4 трэцяга раздзела «Мы, гаспадар, абяцаем павялічваць Вялікае Княства Літоўскае і, што было забрана і адышло, зноў да яго далучыць» [9, с. 45]. Статутам 1588 г. прадугледжавалася асобная для Вялікага Княства Літоўскага сістэма органаў дзяржаўнага кіравання, парадак іх фарміравання. Такім чынам, юрыдычна Вялікае Княства Літоўскае разглядалася як самастойная дзяржава. Гэтая акалічнасць мела значны ўплыў на ўнутрыпалітычнае становішча дзяржавы. Узаемаадносіны паміж Каронай і Княствам мелі шмат супярэчнасцей. Разам з тым, узмацніўся працэс паланізацыі насельніцтва Вялікага Княства Літоўскага. Заключэнне Люблінская унія можа разглядацца ў якасці дакумента, які юрыдычна замацаваў імкненні Польшчы і значнай часткі шляхты Вялікага Княства Літоўскага да паглыблення саюзных адносін. Акт Уніі змяшчаў у сабе асноўныя дзяржаўна-прававыя характарыстыкі новага аб’яднання і прадугледжваў менавіта стварэнне адзінай дзяржавы, таму мы можам лічыць яго дакументам канстытуцыйнага характару, палажэнні якога ўдакладнялі наступныя прававыя акты. Між тым, Унія не вырашала існуючых паміж Польшчай і Вялікім Княствам Літоўскім супярэчнасцей, у першую чаргу тых, якія тычыліся дзяржаўна-прававога стану Вялікага Княства Літоўскага ў складзе Рэчы Паспалітай. ЛІТАРАТУРА 1. Лаппо И.И. Великое княжество Литовское за время от заключения Люблинской унии до смерти Стефана Батория (1569– 1586). Опыт исследования политического и общественного строя. – СПб.: Тип. И.Н. Скороходова, 1901. – Т. 1. – 811 с. 2. Юхо Я.А. Кароткі нарыс гісторыі дзяржавы і права Беларусі: вучэб. дапам. – Мінск: Універсітэцкае, 1992. – 270 с. 3. Грыцкевіч А. Люблінская унія 1569 // Энцыкл. гісторыі Беларусі: у 6 т. / рэдкал.: Г.П. Пашкоў (гал. рэд.). – Мінск: БелЭН, 1997. – Т. 4. – С. 412–413. 4. Акт Люблинской унии 1569 года (текст) [Электронный ресурс]. – URL: https://library.by/portalus/modules/belarus/ readme.php?subaction=showfull&id=1141331680&archive=1291800676&start_from=&ucat=&&ysclid=lo5llfr0nt752926280. (дата обращения: 19.09.2023). 5. Сенат Рэчы Паспалітай // Энцыкл. гісторыі Беларусі: У 6 т. Т. 6. Кніга 1: Пузыны – Усая / Рэдкал.: Г.П. Пашкоў і інш. – Мінск: БелЭн, 2001. – С. 287-289 6. Вяроўкін-Шэлюта У. Пасольская ізба // Вялікае Княства Літоўскае. Энцыкл. У 2 т. — Мінск: Беларус. энцыкл. імя П. Броўкі, 2005. — Т. 2: Кадэцкі корпус – Яцкевіч. – С. 404–405. 7. Канаплёва С. Генрыкавы артыкулы // Вялікае Княства Літоўскае: Энцыкл. У 2 т. – Т. 1: Абаленскі – Кадэнцыя / Рэдкал.: Г.П. Пашкоў (гал. рэд.). – 2-е выд. – Мінск: БелЭн, 2007. – С. 525. 8. Падалінскі У.А. Прадстаўніцтва Вялікага Княства Літоўскага на Люблінскім сойме 1569 года: удзел у працы першага вальнага сойма Рэчы Паспалітай. – Мінск: А.М. Янушкевіч, 2017. – 240 с. 9. Статут Вялікага Княства Літоўскага 1588 г. / Пер. на сучас. бел. мову А.С. Шагун. – Мінск: Беларусь, 2016. – 263 с. Паступіў 02.11.2023 ГОСУДАРСТВЕННО-ПРАВОВАЯ ХАРАКТЕРИСТИКА ЛЮБЛИНСКОЙ УНИИ 1569 ГОДА А.В. ВАЛЕВКО (Полоцкий государственный университет имени Евфросинии Полоцкой) В статье рассмотрены основные положения акта Люблинской унии 1569 года, дана правовая оценка государственной модели Речи Посполитой, согласно с содержанием акта Унии, выявлены особенности государственного устройства Речи Посполитой, проведен анализ научных взглядов на суть и историко-правовое значение Люблинской унии; согласно с положениями акта Унии сделан вывод о политико-правовом положении Великого княжества Литовского в составе Речи Посполитой. Ключевые слова: Люблинская уния, Речь Посполитая, Великое княжество Литовское, сойм. 77 2023 ВЕСТНИК ПОЛОЦКОГО ГОСУДАРСТВЕННОГО УНИВЕРСИТЕТА. Серия D STATE-LEGAL CHARACTERISTICS OF THE UNION OF LUBLIN IN 1569 A. VALEVKO (Euphrosyne Polotskaya State University of Polotsk) The article examines the main provisions of the act of the Lublin Union of 1569; the legal assessment of the state model of the Commonwealth of Independent States is given, according to the content of the act of union; the features of the state system of the Commonwealth of Independent States are defined; the analysis of scientific views on the essence and historical and legal significance of the Union of Lublin was carried out; according to the provisions of the act of union, a conclusion was made about the political and legal status of the Grand Duchy of Lithuania as part of the Commonwealth of Nations. Keywords: Union of Lublin, Commonwealth, Grand Duchy of Lithuania, Diet. 78 

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ARICE – GA No. 730965 Deliverable D8.4 HORIZON 2020 Research and Innovation action Grant Agreement No. 730965 ARICE: Arctic Research Icebreaker Consortium: A strategy for meeting the needs for marine-based research in the Arctic Deliverable 8.4. Quality assurance plan, including guidelines, best practice examples and project handbook © ARICE Consortium Page 1 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 Submission of Deliverable Work Package WP8 Deliverable no. & title D8.4. Quality assurance plan, including guidelines, best practice examples and project handbook Version V2 Creation Date 08.06.2018 Last change 21.01.2019 Status Draft WP lead accepted Executive Board accepted Dissemination level PU-Public PP- Restricted to programme partners RE- Restricted to a group specified by the consortium CO- Confidential, only for members of the consortium Lead Beneficiary Contributors AWI (partner 1) 1 – AWI, 2 – SPRS, 5 – UAF/CFOS, 9 - WOC, 10 – IOPAN, 13 – NERC-BAS, Due date June 2018 Delivery date January 2019 © ARICE Consortium Page 2 of 43 6 – AP, 3 - NPI, 4 - ULAVAL, 7 – CSIC-UTM, 11 – FMI, 8 – CNR, 12 - CNRS, 14 – DTU-AQUA 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 Deliverable No. 8.4. Quality assurance plan, including guidelines, best practice examples and project handbook Abstract D8.4 defines a set of working procedures, processes and best practice guidelines to ensure quality standards of the project outcomes. 1. INTRODUCTION The purpose of this document is to define a consistent set of working procedures, processes and best practice guidelines in order to ensure quality standards of the project outcomes. This document represents a general Quality Assurance Plan (QAP) and project handbook for the ARICE project. Its main aims are To manage the interaction between the project partners during the project lifetime; To check the progress of the work, on a regular basis; To detail how and when the documentation has to be exchanged by the partners and with the European Commission; To set editorial standards for project documents and contents; To provide documents for distribution to the public using the project’s public website available at URL: http://www.arice.eu This document is split into four sections, and includes various annexes: 1. Section I details how the ARICE project is organized and how the relevant consortium bodies will interact during the project; 2. Section II explains how the communication within the Project is organised, and details the publication and communication rules; 3. Section III deals with the technical outputs of the project, including the project reporting; 4. Section IV presents financial statements of the Project In addition to the present QAP, the project will be guided by major reference documents, which define the objectives, the work program and the operational procedures of the ARICE project: - the Grant Agreement (GrA) signed by all beneficiaries - guidance documents provided by the European Commission The Grant Agreement is available on the intranet of the ARICE website for all project partners: https://arice.eu/intranet. © ARICE Consortium Page 3 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 Grant and Consortium Agreements and EC guidance documents can also be found on the EC Participant Portal (EC PP): http://ec.europa.eu/research/participants/portal/desktop/en/home.html. 1.1. Facts about ARICE Acronym/ Contract number: ARICE/ 730965 Title of the Project: ARICE: Arctic Research Icebreaker Consortium: A strategy for meeting the needs for marine-based research in the Arctic Starting date / End date: 1st January 2018 until 31st December 2021 Requested EU contribution: €5,996,563.75 Project Coordinator (PC) and Manager (PM): PC: Nicole Biebow: +49 471 4831 1011, nicole.biebow@awi.de PM: Verónica Willmott: +49 471 4831 2148 Grant Manager: Nancy Lange +49 471 4831 2306 Nancy.Lange@awi.de (in maternity leave until January 2020) and in the meantime substituted by Svenja Hildebrandt: +49 471 4831 2306, Svenja.Hildebrandt@awi.de Scientific officer Agnès Robin: +32 229-93110 agnes.robin@ec.europa.eu 2. PROJECT GOVERNANCE 2.1. General management scheme The general management structure of ARICE is also presented in part B of the DoA. © ARICE Consortium Page 4 of 43 21/01/2019 ARICE – GA No. 730965 2.2. Deliverable D8.4 Project bodies description and responsibilities ARICE is a large project with 14 beneficiaries, two of them non-EU countries (USA and Canada). It therefore requires close cooperation between many institutions and organisations on an international scale. Extensive managerial efforts are required to: - Ensure that all beneficiaries perform the duties assigned in the GrA Annex I, - Ensure that all deliverables/milestones/results are handed in in time and that each partner has sufficient resources, - Detect and help to solve problems, which may arise due to insufficient communication between beneficiaries or for any other reason. From a Quality Assurance (QA) point of view, it is important that the project management at all levels of the project (strategic, executive and operational), is high skilled and experienced in its duties. The following paragraph describes the responsibilities of each entity. The responsibilities and composition of the different project governance bodies are described in detail in the ARICE CA and the DoA. The following compilation in table 1 is only a summary of the detailed description in these documents: Table 1: ARICE governance bodies Body Composition/Responsibilities Decision approval Planned meetings Project Coordinator (PC) and Management Team (MT) The PC of ARICE is Dr. Nicole Biebow, Head of the International Cooperation Department at AWI. She has extensive experience in the management and coordination of large international consortia. The PC will be supported by the Management Team (MT) consisting of four persons based at AWI. The MT is led by Dr. Verónica Willmott (AWI) who has proven experience in project and calls for proposals management. Verónica Willmott will work as a project manager (PM) for ARICE and is tasked by the PC with the day-to day management of the project. By GA and EB N/A She will be assisted by the responsible EU grant manager at AWI (Nancy Lange /Svenja Hildebrandt). The PC supported by the MT will manage the entire Consortium ensuring ARICE’s progress and the achievement of all results envisaged in this proposal. The PC will make all day-to-day decisions and will be the intermediary between the European Commission (EC) and the consortium in all matters. She will be the primary representative of the project in dealings with the EC, policy makers, the public media and other organisations. The PC will task the PM to initiate and organise the production of all reports requested by the EC. © ARICE Consortium Page 5 of 43 21/01/2019 ARICE – GA No. 730965 Steering Board (SB) The fundamental role of the Steering Board (SB) is to ensure successful execution of the project. The SB reports to and is accountable to the GA. It will consist of the PC, MT, and WP leaders. Chaired by the PC, the SB will meet at least quarterly, or more often as required by the course of the project. Any member of the SB can call for an extraordinary meeting by a written request. Deliverable D8.4 By GA Quarterly Self-decision making organisation Every year The SB’s main responsibilities include the delivery of the project work plan, review of the project progress and the resources status, ensuring the execution of the risk management plans of the project if necessary and ensuring the smooth internal cooperation and relationship between consortium members as well as external project stakeholders. The meetings will be scheduled if possible in conjunction with other meetings and video- and teleconferencing will be used as often as possible to reduce the travel costs. General One senior representative from each partner of the project Assembly (GA) forms the GA, the ultimate decision-making body of the consortium. The GA meets at least once a year, over the duration of the project. The GA is chaired by the PC. One permanent representative of the Advisory Board will assist the GA in its decisions but has no own voting rights. Extraordinary meetings of the GA can be convened by a written request from the Steering Board or 1/3 of the members of the GA. The GA´s responsibilities include the strategic planning of the project ARICE, the approval of the project deliverables as defined in the Grant Agreement, the approval of periodic and final reports to the EC, the review of the project progress against milestones, and monitoring and implementing any changes necessary in the Consortium Agreement. Scientific Liaison Panel (SLP) The Scientific Liaison Panel (SLP) is comprised of internationally recognised external experts in the fields of Arctic marine research. The members of the SLP are identified by WP4 and appointed by the GA. One Ethical Advisor has been appointed as member of the Scientific Liaison Panel to oversee any potential ethical issue arising in the evaluation. Self-decision At least 2 making (one per organisation. call) Funding decisions to be approved by GA The role of the SLP is to assist ARICE in the evaluation process and to act as liaison of the ARICE project and the scientific community, maintaining communication and © ARICE Consortium Page 6 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 coordinated actions with research and other marine organisations. The chair of the SLP will be elected from the board and recommended to the GA for approval. Meetings of the SLP will take place to perform a consensus evaluation of the proposals. The Chair of the SLP will take part on the GA meetings and advise on scientific issues. Operational Liaison Panel (OLP) The Operational Liaison Panel (OLP) is comprised of operators of European PRVs and international icebreakers. The members of the OLP are identified by WP1 as the OLP will have a key role on the harmonisation of the Arctic fleet. The members of the OLP are appointed by the GA. The Chair of the OLP will be elected from the panel and recommended to the GA for approval. Self-decision At making convened organisation. meetings, as needed per WP The operators of the six ARICE PRVs within the OLP will also participate at the logistic evaluation of the proposals submitted to the ARICE calls for proposals. Industry Liaison Panel (ILP) The Industry Liaison Panel (ILP) is comprised of Self-decision representatives of the Arctic maritime industry. The role of making the ILP is to stimulate the connection between science and organisation industry and to give recommendations and support to the strategic vision of ARICE on scientific cooperation with industry. The members of the ILP are nominated by WP2 and appointed by the GA. The chair of the ILP will be elected from the board and recommended to the GA for approval. Advisory Board (AB) The Advisory Board (AB) is comprised of international recognised experts in the field of Arctic marine research, representatives of related Polar organisations (e.g. EPB) and relevant stakeholders from industry and politics. The members of the AB are identified by the SB and appointed by the GA. The AB will advise the GA and SB on the enhancement of relationships between Arctic marine industry/Arctic marine research. It will be fundamental in stimulating connection with international partners and organisations as well as with other European initiatives and projects. Its role is also to ensure the transparency of the evaluation process, avoid any conflict of interests and helping ARICE to deal with ethical issues by putting in place the procedures to handle them appropriately. At convened meetings, as needed per WP Self-decision making organisation The chair of the AB will be elected from the board members and recommended to the GA for approval. One member of the AB will take place at the SLP proposal evaluation meeting to ensure the transparency of the evaluation process. Meetings of the AB will take place adjacent to the © ARICE Consortium Page 7 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 yearly GA to give the AB members the possibility to follow project progress most closely and to be directly involved in the discussions leading to strategic decisions. The SLP and the ILP will seek the advice of the AB whenever it is necessary for the project course. They can call for extraordinary AB meetings or consult the AB members by video-or teleconferences and electronic communication. WP Leaders While the MT has the overall responsibility for the execution of the work plan, the WP leaders, in conjunction with the appointed task leaders, conduct and manage the project activities. They collaborate closely, using a system of regular internal reporting. At least every third month, task leaders shall summarise their progress towards project deliverables to the WP Leaders, who will review the activity against the work plan and, following discussion with the task leaders, consider if interim targets or measures are required. These reviews will also serve as the basis of more formal reports for the SB, PC, GA and, ultimately, the European Commission. By SB When needed Task Leaders The task leaders are responsible for the implementation of the individual tasks of the work plan and shall report the task progress to the WP leaders. Task leaders shall establish interactions between individual partners of the task by arranging individual task meetings (videoteleconferenced/web-based virtual meetings, if appropriate). They are also responsible for establishing and maintaining links to other tasks as necessary. By WP leaders and SB When needed Each consortium body shall not deliberate and decide validly unless a quorum of two-third (75%) of its members are present or represented, as specified in the CA (article 6.2.3). 2.3. Work Package and Task management The management of the WPs and a frequent interaction between WP and task leaders is one of the critical aspects of the success of ARICE. The project is divided into 9 work packages including several tasks which are closely interrelated under a coordinated management scheme (WP8) (see table 2). © ARICE Consortium Page 8 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 Table 2: ARICE Work Packages and Tasks. Work Package / Task Leaders WP1 Towards an Arctic Research Icebreaker Consortium SPRS Task 1.1 Improving the coordination of European PRVs Task 1.2 Alignment of national research plans Task 1.3 Towards an International ARICE Task 1.4 Model for cooperation of the International ARICE WP2 Establishing a regular dialogue with the maritime industry WOC Task 2.1 Arctic science community/industry dialogue WOC Task 2.2 Identification of joint priorities for Arctic observations Task 2.3 Opportunities for key technologies and innovation WOC WP3 New generation of polar researchers and professionals AWI (APECS) Task 3.1 Online training and resources for multiple audiences Task 3.2 In person/on-site training courses Task 3.3 Assessment of training activities AWI (APECS) WP4 Proposal management and shared evaluation AWI Task 4.1 Preparation of call for proposals for ship-time Task 4.2 Implementation and dissemination of the call for proposals Task 4.3 Evaluation and selection of proposals Task 4.4 Allocation of ship-time and post-cruise assessment WP5 Transnational access to the Arctic Ocean CSIC-UTM Task 5.1 ARICE cruises CSIC-UTM WP6 Expanding the monitoring and observation capacities in the Arctic Ocean FMI Task 6.1 Survey of existing automatic ship based observations Task 6.2 “Programme of ships and platforms of opportunity” © ARICE Consortium Page 9 of 43 SPRS DTU Aqua NPI SPRS SPRS DTU Aqua SPRS AWI CNR WOC IOPAN AWI (APECS) AWI (APECS) CNR AWI CSIC-UTM AP AWI AWI IOPAN CSIC-UTM AWI FMI DTU-Aqua WOC FMI 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 FMI Task 6.3 New technological solutions for data collection WP7 Enhancing virtual and remote access to data Task 7.1 Data management requirements Task 7.2 Data Management System Design Task 7.3 3D Virtual Icebreaker WP8 Management of the Consortium AWI Task 8.1 Contractual and financial management AWI Task 8.2 Operational management AWI Task 8.3 Communication and Outreach AP Task 8.4 Evaluation of the impact of ARICE CNR WP9 Ethics requirements AWI Task 9.1 Ethics requirements AWI CSIC-UTM AP AP CSIC-UTM AP FMI AP NERC-BAS More information about the ARICE WPs and tasks can be found in the DoA. 2.4. Meetings Project meetings necessary for the success of the ARICE project. They are needed to maintain relationship, to promote information exchange, to make agreements and to take major decisions. All beneficiaries have to participate in the GA meetings once a year; since this is the only occasion at which the whole consortium meets. Liaison panels and advisory board representatives are invited to participate in the GA meetings but have no voting rights. The consortium has decided to have meetings of the ARICE governance bodies with the following minimum frequency: − General Assembly: once a year − AB Meetings: once a year in association with the General Assembly − Steering Board: at least every 3 months, preferably by video- or teleconference − Work Packages or Tasks: on request Additional face two face meetings may take place as required but should be limited and video conferences (VC) will be preferably used in order to follow the work in progress. Meetings will be grouped together whenever possible in order to accelerate communication and avoid unnecessary trips and carbon waste. 2.4.1. Official meetings GA meetings © ARICE Consortium Page 10 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 One meeting every year. The planned meetings for 2018, 2019 and 2020 are the following: # Date/Period Type of Meeting Meeting Place 1 05/02/201807/02/2018 Kick-Off Meeting (KOM) and 1 General Assembly 2 28-29/03/2019 2nd General Assembly rd rst Bremerhaven, Germany Lisbon, Portugal 3 tbd 2020 3 General Assembly tbd 4 tbd 2021 4th General Assembly tbd Executive Board meetings At least one meeting every 3 months. The next meetings for 2018 have been planned as follows: # Date/Period Meeting place Special agenda item Parallel event 1 23/03/2018 VC Terms of Reference of EB, Call for shiptime proposals, nominations to SLP 1rst GA 2 29/06/2018 VC WP progress and implementation of Liaison panels / 3 October 2018 VC Information on the evaluation of proposals, implementation of the advisory board, pending deliverables / 4 December 2019/ January 2019 VC tbd / 2.4.2. Other meetings Work Package / Task Meetings Technical meetings should take place within each WP or even task. The periodicity is not contractually defined, although an average of one meeting per year is intended for each WP usually in association with the ARICE GA. Attendance at external meetings Meetings with scientists, industry, Arctic research vessels operators and stakeholders are an important part of the ARICE project since we need their collaboration to improve the access to research vessels in the Arctic Ocean. ARICE will try to implement most of its workshops at international conferences with a large stakeholder representation, to avoid additional travel and to facilitate workshop participation. The ARICE MT will take care that all events are announced early in advance and to a wide audience. Project partners of ARICE who attend a meeting on behalf of the project are asked to report back to the consortium about the outcomes and major action items derived from this © ARICE Consortium Page 11 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 meeting. Their reports will be made available to the consortium on the internal ARICE website. A template for reporting is available on the Internal Webpage and in Annex II.10. 2.4.3. Meeting organisation Official (GA and SB) GA and SB meetings shall be announced early in advance to allow all project partners to be present or represented. The minimum number of days preceding the meetings are 45 for GA and 14 calendar days for SB. Responsible and schedule Agenda Produced by the MT. Distributed 21 calendar days prior the meeting to the project partners. Is considered to be final if no comments are received within one week after its distribution. Actions item list The actions item list is the summary of the main managerial, administrative and technical decisions taken during the meeting. It is produced by MT. Distributed within two weeks after the meeting to the project partners. Is considered to be final if no comments are received within one week after its distribution. Minutes The minutes summarise the discussions and decisions of the GA or EB. They are produced by MT. They will be sent out at the latest 30 days after the meeting. The minutes of the SB are sent to the GA for information. Are considered to be final if no comments are received within two weeks after their distribution. Other meetings (Work Packages, tasks, etc…) Responsible and schedule Agenda Produced by the organising partner or WP leader. Distributed two weeks prior the meeting to the involved partners and to the MT. Is considered to be final if no comments are received within 2 days prior its distribution. Minutes Produced by the organising partner or WP leader. Distributed within two weeks after the meeting to the involved partners and to the MT. Are considered to be final if no comments are received within two weeks following their distribution. © ARICE Consortium Page 12 of 43 21/01/2019 ARICE – GA No. 730965 © ARICE Consortium Page 13 of 43 Deliverable D8.4 21/01/2019 ARICE – GA No. 730965 3. Deliverable D8.4 PROJECT COMMUNICATION 3.1. Main communication tools Various tools will be used to allow and foster internal and external communication (see Table 3). Table 3: Tools for internal and external communication. Used tools Purpose/comment Requirements Public project website (www.arice.eu) for presenting the project and its outcome to the public. All information and documents on this website are public by nature. Social Media ARICE is active in three social media channels: Twitter and Facebook. Twitter is used to share information with the general public, journalists, scientists and policy makers on ARICE news and events. The Facebook group provide a platform for discussions with group members. Needs to be administered Frostbites ARICE will create teaching/training materials as a legacy from the summer school. A dedicated software is needed Project internal website Official document repository: data storage for all documents produced by the project (deliverables, reports, minutes, presentations etc.) or for all documents necessary for the technical, financial and contractual follow-up (Deliverables follow-up, budget breakdown, amendment request etc.) Login by institutional account, with password/login External Public website Internal Exchange of project material (templates, official and contractual documents e.g. reports, meeting materials, financial documents, drafts, others) Project materials & documentation exchange should be made preferably via the internal website Meeting organization (agenda, minutes, presentations) Working area of the project E-mail The internal website contains mailing lists for the different project bodies of ARICE. Limited exchange of project materials Emails should only be sent to those mailing lists which are directly related to the subject of the email to avoid a snowball effect. The relation to ARICE shall be made clear in the subject. Avoid proliferation Large Attachment should preferably be stored on the internal website and not send by email. © ARICE Consortium Page 14 of 43 21/01/2019 ARICE – GA No. 730965 Video Conference (VC) 3.2. Deliverable D8.4 VC should be used for short meetings in smaller groups when and if possible. This reduces travel costs and accelerates communication. All required documents must be distributed before the meeting via the collaborative site/email. ARICE external communication The ARICE public website is available at the URL: http://www.arice.eu. It is administered by Arctic Portal and AWI has also administration rights. The website is based on Joomla. It plays an important role in ARICE, as it is the focal tool for communicating and disseminating information generated by the consortium partners. Potential end-users will be scientists, industry, decision-makers, stakeholders and the general public. In order to facilitate the fast exchange of information among consortium partners and to update the website several templates were developed (see Annex II). Whenever an activity is completed (deliverable; attendance to a meeting; publication…) project beneficiaries are requested to fill them in and submit to the Management Team. With this information the portal will be constantly updated with deliverables, presentations, news, events, etc.. Other important means of communication for ARICE are the social media channels Twitter and Facebook. Both channels are used for outreach and engagement roles. A e-newsletter shall be released and distributed by email to inform on news and events. Interested users can register on the public website to receive the ARICE e-newsletter by filling in a registration form, where they can record their e-mails. Users have the possibility to unsubscribe the e-newsletter at any time. 3.3. Internal project website The internal project website gives access to the whole ARICE consortium through a user login/password. The login to the ARICE internal website can be found at the following address: https://www.arice.eu/intranet © ARICE Consortium Page 15 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 3.3.1. Structure of the internal website The intranet of ARICE offers different sections with important documents for the ARICE consortium. Every consortium partner has got a login key and a password to access the internal website. The intranet is divided in two sections: 1) Documents The folder “documents” contains: - The grant agreement - Mailing lists of project participants - Presentations and promotional material, and - Diverse templates The different sections will be continuously updated during the life-time of the project. © ARICE Consortium Page 16 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 2) Deliverables The section “deliverables” shows a table with all the deliverables from the project ARICE in final version as submitted to the EC. Submitted deliverables are downloadable in pdf. © ARICE Consortium Page 17 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 3.3.2. Access rights The ARICE internal website is accessible by all ARICE partners but only the following website administrators (Verónica Willmott (AWI), Ævar Karl Karlsson (Arctic Portal) and Fanney Sigrún Ingvadóttir (Arctic Portal) can upload documents and create texts. 3.3.3. Mailing lists To facilitate internal communication within the project, a general mailing list and dedicated mailing lists for every WP and tasks have been created and are maintained up-to-date by the MT. The general rules set for e-mails (see above) also apply to mailing lists. If an update in a list is required, send an email to the MT with the correct e-mail address and targeted group. 3.4. Document and image management 3.4.1. Document confidentiality All ARICE documents are considered as “consortium confidential”, except when they are explicitly mentioned as public documents (PU) in the DoA. 3.4.2. Document templates All document templates can be found on the internal site in the “Documents” folder from the intranet. In all document templates, the Author is the lead beneficiary responsible for the document delivery. The following templates are available (see Appendix for examples): For official correspondence: An official paper letter (Word format): only on request to the MT For communication: A template for publications (Word format) A template for dissemination activities (Word format) A template for relevant news (Word format) A template for news on a Deliverable (Word format) For general purposes: General Purpose Document (Word format) Presentation Template (Power Point format) © ARICE Consortium Page 18 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 For meetings: Meeting Agenda (Word format) Meeting Minutes (Word format) For reporting: Deliverable Template (Word format) Milestone Template (Word format) If needed, this list can be updated during the Project life. 3.4.3. Naming conventions and versioning of documents Recommendations related to the document editing are: − All documents have to be produced in Microsoft Word format for working versions and Acrobat PDF for final versions; − Editing language should be set to UK English. To facilitate their identification, documents produced within the project (Deliverable, Milestone, report, presentation, minutes, paper etc …) shall integrate the naming conventions defined in Annex III. For the versioning, the documents should be simply implemented as following: 1, 2, 3… 3.5. Publications and dissemination of Project knowledge Publications and dissemination activities must be reported to the EC in each periodic report and in particular in the final report via dedicated areas in the EC PP. The list of publications and dissemination activities is maintained up-to-date by the Communication leader (Arctic Portal) during the project life using information provided by project’s partners (see § 4.2.2.2 and dedicated templates in Annexes II.2 and II.3). 3.5.1. Types of publications and dissemination activities Publications or communications may be wholly or largely based on work done for the project and/or contain some material based on work done in the project. In agreement with the classification described on the EC Participant Portal (PP) (see § 4.2.2.2 and the dedicated template in Annex II.2), the following presentation modes are included under the term “publication”: a peer reviewed publication, a paper in Proceedings of a Conference or a Workshop, an article or a section in an edited book or book series, a Thesis, or a University publication. The other presentation modes included under the term “Dissemination activities” (see §4.2.2.2 and the dedicated template in Annex II.3) are: Organisation of a Conference or a Workshop, websites or applications, press releases, flyers, articles published in the popular press, videos, media briefings, presentations, oral presentation to a wider public or to a scientific event, exhibitions, thesis, interviews, films, TV clips or posters. © ARICE Consortium Page 19 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 3.5.2. Rules for publications and dissemination activities, and approval procedure Usual intellectual ownership and decency rules should be applied. Any proposed publication or communication, regardless of the media (including any plan or model), of the partner's own knowledge is required to be submitted to the other interested partners and to the European Commission. To this end, a brief description and the subject of the proposed publication or communication shall be submitted to the other project partners. The partners shall have the right to object to the publication in accordance with Article 29.1 of the GrA. For all types of publications and dissemination activities listed in § 3.5.1, beneficiaries must fill in the appropriate templates (available in Annexes II.2 or II.3) and send them by e-mail to the MT. For publications and communication to external events in particular, the procedure for approval is the following: The author(s) send(s) the template for publications (see Annexe II.2) or the template for dissemination activities (see Annexe III.2) duly filled in 3 or 6 weeks prior to the date of publication or submission to the MT. The author(s) send(s) the publication/communication content to all interested beneficiaries for comments or approval. When relevant, the author(s) transmit(s) the final version to the MT for storage on the projects internal website, and the MT further to the Communication leader for publication on the ARICE public website. In addition, it is mandatory for the author(s) to include in publications appropriate acknowledgement to the EC as follows: These results have been achieved within the ARICE project funded by the European Union’s Horizon2020 research and innovation programme under grant agreement n°730965. When referring to contributions from other partners, the author(s) is(are) free to phrase the acknowledgement, but must include names, institutes and a reference to the ARICE project such as the Internet website address (http://www.arice.eu). © ARICE Consortium Page 20 of 43 21/01/2019 ARICE – GA No. 730965 4. Deliverable D8.4 PROJECT DELIVERY AND PROJECT REPORTING The project deliverables are split into 2 categories: Deliverables and Milestones – see 4.1 Periodic and final reports – see 4.2 Deliverables are the outputs of the tasks within the Work Packages and will be submitted to the EC by the MT via the EC PP. (http://ec.europa.eu/research/participants/portal/desktop/en/home.html) 4.1. Deliverables & Milestones Deliverables and milestones shall be produced according to schedule described in 4.1.1. The following reporting recommendations should be taken into account: Quality standard (see 4.1.2) Approval process and storage (see 4.1.3) Follow-up (see 4.1.4) 4.1.1. Deliverables and Milestones schedule Deliverables and milestones are to be delivered by the project and assessed at the Due Date (DD) indicated in the DoA. Due Dates (last working day of calendar month) and responsible beneficiaries of deliverables and milestones are respectively listed in the DoA. 4.1.2. Quality standard The primary responsibility of Work Package (WP) leaders, Task leader and MT is to establish and maintain high standards of technical and professional quality with regard to the objectives set for the project. All deliverables and milestones in ARICE need to be approved by the Steering Board (SB) prior to uploading them to the EC PP or publishing them. All deliverables should be delivered with a summary report and according to the following table and associated guidelines. Deliverables Technical specifications and Quality standards Deliverable report Shall contain a detailed description of the technical aspects of the deliverable. The template is available on the internal website. See Annex III for the naming convention. Shall be accompanied with an Executive Summary to be provided in the “Template for news on Deliverables” available in Annexe II.3, in order to facilitate its communication to the project’s partners and to public audience via the ARICE website. © ARICE Consortium Page 21 of 43 21/01/2019 ARICE – GA No. 730965 Milestone report Deliverable D8.4 Assessment shall be formalised in a milestone assessment report. The template is available on the internal website. See Annex III for the naming convention. The milestone report shall include: The purpose of the milestone, the status, the assessment criteria and the consequences of the milestone result. The author(s) should comply with general recommendations given for scientific and technical reporting and publishing, and specifically verify that: − Reference to public sources of information are given; − Only persons who significantly contributed to the project and manuscript preparation are listed as co-authors; − The publication do not contain speculative opinions, although it can use scientific evidence to challenge current concepts or propose new ideas that will encourage progress and discussion; − It is free of evident commercial or private interest, but must neither obscure proper names when they are required. In the case of critical time delays, and in collaboration with the WP Leaders, measures should be proposed in an effort to avoid potential problems. These measures could be: 1. Re-organisation and re-deployment of the project time plan. All affected partners should be informed about the delay. It is of particular importance to try to minimise resulting project execution delays especially in case of tasks comprising key deliverables of the project; 2. Re-allocation of scientific resources; 3. Although not recommended, request of extension of time to the EC. In this case, the project coordinator presents a detailed report with the difficulties and drawbacks that led to violation of the time constraints and the measures taken during the project in order to overcome these problems as well as the partners responsible for the time delays. 4.1.3. Process for Deliverable/Milestone delivery and storage The process to ensure a proper delivery of the deliverables and milestones at the Due Date is presented hereafter. The deliverable/milestone approval is based on a 6-step process described as follows: − One month before the DD, the MT sends a reminder to the deliverable/milestone leader, copy to the WP and Task leaders; − Two weeks before the DD, the deliverable/milestone leader sends the deliverable to the WP Lead for validation; − At the DD, the deliverable/milestone leader sends the deliverable/milestone to the MT, copy to the WP leader. It has to be accompanied by the “Template for news on Deliverable” (see annex II); the MT will inform the SB and ask for approval by the SB; © ARICE Consortium Page 22 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 − The MT uploads the final and approved version to the intranet site. − The MT submits the approved deliverable to the EC via the EC PP, and informs the WP leader about the submission. For a milestone, the MT updates the Milestone table on the EC PP; − For public deliverables, the MT will take care that the deliverable is published on the project’s public website and announced widely. 4.1.4. Deliverables and Milestones follow-up The follow-up of deliverables and milestones is carried out using updated sheets available on the internal website. 4.2. Periodic and final reporting 4.2.1. Periodic reporting (M18, M36, M48; M60) This process presented hereafter only deals with technical aspects, please refer to § 5 for financial aspects. Periodic reports have to be submitted to the EC 60 days after the end of the reporting period. ARICE spans three reporting periods: Period 1: M01-M18: 01.01.2018 - 30.06.2019 Period 2: M19-M36: 01.07.2019 - 31.12.2020 Period 3: M37-M48: 01.01.2021 - 31.12.2021 4.2.1.1. Content and structure A template for the periodic technical reports will be created according to the H2020 online manual, and this template will be uploaded to the internal ARICE website. The periodic technical report will include an explanation of work carried out, an overview of progress, a publishable summary and a questionnaire. Layout and content of the periodic report must comply with the instructions and guidance notes established by the EC (http://ec.europa.eu/research/participants/docs/h2020-funding-guide/grants/grantmanagement/reports/periodic-reports_en.htm ). The periodic technical reports are prepared using a dedicated interface available on the EC PP and are structured as follows: The explanation of the work carried out and the overview of the progress should show how the action is being implemented and what has already been achieved (as compared to the objectives, milestones and deliverables described in Annex 1 of the DoA). The PC will check if all deliverables due for the period have been submitted. If work planned was not carried out, the beneficiaries must explain why. The overview of the progress must also describe how achieved results are exploited and disseminated. The publishable summary must give a brief description of the action, presenting its objectives and the results achieved (in an ‘easy to read’ way, understandable for a nonspecialist audience).The summary must be fit for publication, so that the Commission can publish it on its website right away. If needed, the Commission may make changes to the © ARICE Consortium Page 23 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 summary and publish it (after having given the coordinator the opportunity to comment). The PC must ensure that none of the material submitted for publication includes confidential or ‘EU classified’ information. The questionnaire must be filled out to provide the Commission with regular up-to-date information for monitoring the action (and ultimately the Horizon 2020 Framework Programme). The questionnaire consists in structured information on: - performance indicators (defined in Annex II to the Horizon 2020 Specific Programme) - information to monitor the implementation of Horizon 2020 on ‘cross–cutting issues’ (see Annex III to the Specific Programme) and to assess the progress of Horizon 2020 against the objectives defined for the ‘societal challenges’ (see Article 3 and Annex I to the Specific Programme). It is designed in a modular way, consisting as much as possible of structured questions, by topic (e.g. publications, patents, innovation, etc.). 4.2.1.2. Approval process for periodic reporting: technical aspects The following process has been set up to ensure a proper delivery of periodic reports within 60 days after the Due Date. The document is elaborated through a multi-level process where key contributors report according to the template provided by the EC where technical and financial aspects shall be reviewed (see 4.2.1.1). − One month before the end of the period, the MT sends the template for the periodic technical report to the WP leaders for completion of the chapter work progress and achievements per WP during the period; − 15 days after the end of the period, WP leaders transmit their contribution to the MT for consolidation of all WP contributions; − 30 days after the end of the period, the MT starts the preparation to the publishable Summary and transmits the final version to the EB 45 days after the end of the period; − 55 days after the end of the period, the SB validates the final version of the Publishable Summary; − 60 days after the end of the period, the MT submits the periodic report to the EC portal and stores its main components on the internal website. 4.2.2. Final reporting (M60) including dissemination activities and exploitation followed up during the project life In addition to the periodic report for the last reporting period, the coordinator must submit the final report within 60 days following the end of the last reporting period. The final report must include the following: (a) a ‘final technical report’ with a summary for publication containing: (i) an overview of the results and their exploitation and dissemination; (ii) the conclusions on the action, and © ARICE Consortium Page 24 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 (iii) the socio-economic impact of the action; (b) a ‘final financial report’ containing (i) a ‘final summary financial statement’, created automatically by the electronic exchange system, consolidating the individual financial statements for all reporting periods and including the request for payment of the balance and (ii) a ‘certificate on the financial statements’ for each beneficiary, if it requests a total contribution of EUR 325 000 or more, as reimbursement of actual costs and unit costs calculated on the basis of its usual cost accounting practices (see Article 5.2 and Article 6.2, Point A). Content and structure of these two documents, in particular the final report are described hereafter. Procedures for the preparation and the approval of these two documents will be discussed and approved during the last General Assembly of the project in 2021. 4.2.2.1. Structure of the project final report The final technical report is a summary for publication that should present an overview of the results, their exploitation and dissemination, the action’s conclusions and its socioeconomic impact. A template for the final report will be created as soon as the H2020 Online Manual is updated and detailed instructions for the structure of the final report are available. The template will be uploaded to the internal ARICE website. Layout and content of the final report must comply with the instructions and guidance notes established by the EC. It is prepared using the dedicated interface available on the EC PP and is structured as follows: The final publishable summary must cover the whole action. Like the summaries for the periodic reports, the final summary must be written in an ‘easy to read’ way and be understandable for a non-specialist audience. The coordinator must ensure that none of the material submitted for publication includes confidential or ‘EU classified’ information. The summary should include: - an up-to-date link to the action’s website - project logos, diagrams, photographs and videos illustrating the work of the action (if available) It may also include a list of all beneficiaries, with contact names (if this information should be published). 4.2.2.2. Update of the list of publication and dissemination activities within the EC Participant Portal (EC PP) It is mandatory to report on publications and dissemination activities via the EC PP through two dedicated links accessible in the EC PP by clicking on the RD icon. Areas for publications and for dissemination activities are maintained up-to-date in the EC PP by the Communication leader using information provided by the project’s beneficiaries in appropriate templates (see § 3.5.2 for the procedure and Annex II.2 for the template on Publications, and Annex II.3 for the template on Dissemination activities). © ARICE Consortium Page 25 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 − Main page for publications to be filled in using information from the template for publications available in Annexe III.1 − Main page for dissemination activities to be filled in using information from the template for publications available in Annexe III.2 4.2.2.3. Update of the list of exploitable results and project foreground within the EC PP It is mandatory to report on publications and dissemination activities via the EC PP through two dedicated links accessible in the EC PP by clicking on the RD icon. 4.2.3. Transmission of periodic and final reports The Coordinator on behalf of the Consortium shall transmit the above documents to the EC via the EC PP. Reports submitted to the EC, in particular their publishable parts, shall be of a suitable quality to enable direct publication without any additional editing. By submitting the publishable reports to the EC, the MT will also check that they do not include any confidential material. © ARICE Consortium Page 26 of 43 21/01/2019 ARICE – GA No. 730965 5. Deliverable D8.4 FINANCIAL ASPECTS 5.1. Pre-financing, interim and final payments As per the ARICE GrA, the project received a pre-financing of 3,198,167.33€ (representing 53,33% of the ‘maximum grant amount’ of 5,996,563.75€), including: ⇒ An amount of EUR 299,828.19 corresponding to 5% of the maximum grant amount (see Article 5.1),is retained by the Commission from the pre-financing payment and transferred into the ‘Guarantee Fund’. ⇒ 2,898,339.14 € paid to the Coordinator as a first advance payment. Following the payment by the EC, the Coordinator distributed this amount to the project’s beneficiaries. Interim payments reimburse the eligible costs incurred for the implementation of the action during the corresponding reporting periods. The Commission will pay to the coordinator the amount due as interim payment within 90 days from receiving the periodic report (see Article 20.3), except if Articles 47 or 48 apply. Payment is subject to the approval of the periodic report. Its approval does not imply recognition of the compliance, authenticity, completeness or correctness of its content. The total amount of pre-financing and interim payments must not exceed 90% of the maximum grant amount. The final payment will be made within 30 days after approval by the EC of the final reporting, and the final distribution of the EC financial contribution will be described and transmitted to the EC via the final financial report. 5.2. Cost monitoring and financial reporting via the EC Participant Portal (EC PP) 5.2.1. Eligibility criteria Eligible costs are defined in Article 6.1 of the GrA (General conditions) and further details are provided in the H2020 Online Manual available on the internal website or on the EC PP (http://ec.europa.eu/research/participants/data/ref/h2020/grants_manual/amga/h2020amga_en.pdf#page=160) Eligible costs have to meet the following criteria: − Actual : real costs, − Incurred by the beneficiary, − Incurred during the duration of the Project (01/01/2018 – 31/12/2021), with the exception of costs relating to final reports and certificates on the financial statements, − Determined according to the usual accounting and management principles and practices of the beneficiary identifiable and verifiable, − Used for the sole purpose of achieving the objectives of the Project and its expected results, in a manner consistent with the principles of economy, efficiency and effectiveness, © ARICE Consortium Page 27 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 − Recorded in the accounts of the beneficiary and, in case of any contribution from third parties, recorded in the accounts of the third parties, − Indicated in the estimated overall budget annexed to the DoA. 5.2.2. Direct and indirects costs For further details on Direct and Indirect costs, please refer to the EC Financial guide (see § 5.2.1) The reimbursement of beneficiaries shall be based on their eligible direct and indirect costs. Direct costs are all those eligible costs which can be attributed directly to the project and are identified by the beneficiary as such, in accordance with its accounting principles and its usual internal rules. The following direct costs may be considered eligible (this list is not exhaustive): − The cost of personnel assigned to the project; − Travel and subsistence allowances for staff taking part in the project; − The costs of consumables and supplies provided they are identifiable and assigned to the project; − Certificate on the methodology and certificate on the financial statements; − Conference fees. Beneficiaries must keep all original support documents for 5 years after the end of the Project, i.e): The beneficiaries must keep the records and documentation supporting the costs declared (GrA article 18.1), in particular the following: (a) for actual costs: adequate records and other supporting documentation to prove the costs declared, such as contracts, subcontracts, invoices and accounting records. In addition, the beneficiaries’ usual cost accounting practices and internal control procedures must enable direct reconciliation between the amounts declared, the amounts recorded in their accounts and the amounts stated in the supporting documentation; • Invoices (hotel, restaurant, purchases, etc.), • Tickets (train, flight, subway, bus, etc.), • List of presence signed by the participants to a meeting + programme, • Other cost justifications. (b) for unit costs: adequate records and other supporting documentation to prove the number of units declared. For trans-national access to research infrastructure: This documentation must include records of the names, nationalities, and home institutions of users, as well as the nature and quantity of access provided to them. © ARICE Consortium Page 28 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 Beneficiaries do not need to identify the actual eligible costs covered or to keep or provide supporting documentation (such as accounting statements) to prove the amount per unit. In addition, for unit costs calculated in accordance with the beneficiary’s usual cost accounting practices, the beneficiaries must keep adequate records and documentation to prove that the cost accounting practices used comply with the conditions set out in Article 6.2 AGA. The beneficiaries, and linked third parties, may submit to the Commission, for approval, a certificate (drawn up in accordance with Annex 6) stating that their usual cost accounting practices comply with these conditions (‘certificate on the methodology’). If the certificate is approved, costs declared in line with this methodology will not be challenged subsequently, unless the beneficiaries have concealed information for the purpose of the approval. In addition, in case of audits, time-sheets for personnel costs justification must be kept for 5 years from the end of the Project: In addition, for personnel costs (declared as actual costs or on the basis of unit costs), the beneficiaries must keep time records for the number of hours declared. The time records must be in writing and approved by the persons working on the action and their supervisors, at least monthly. In the absence of reliable time records of the hours worked on the action, the EC may accept alternative evidence supporting the number of hours declared, if it considers that it offers an adequate level of assurance. As an exception, for persons working exclusively on the action, there is no need to keep time records, if the beneficiary signs a declaration confirming that the persons concerned have worked exclusively on the action. Indirect costs, also called overheads, are all those eligible costs which cannot be identified by the beneficiary as being directly attributed to the project, but which can be identified and justified by its accounting system as being incurred in direct relationship with the eligible direct costs attributed to the project. The indirect cost rate for all beneficiaries within ARICE is 25% of the total direct costs (excl. subcontracting). 5.2.3. Financial Reporting by each beneficiary at M18, M36, M54 and M60 Each beneficiary must declare the costs incurred during the reporting period and must submit them to the EC via the Coordinator within 60 days after the end of the reporting period (see § 4.2.1). This financial statement has to be filled in using a web-based tool accessible from the EC PP, and all financial statement forms and documents shall respect the guidelines provided by the EC (see § 5.2.1). Each beneficiary remains responsible to the EC for its costs claimed even after payment by the EC and even after submission of a Certificate of Financial Statement (CFS). 5.2.3.1. Preparation of the Financial Report by each beneficiary Costs incurred by the beneficiaries during the reporting periods are declared at the end of each period, i.e. M18, M36 and M48. In order to prepare the cost statement each © ARICE Consortium Page 29 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 beneficiary has to create a draft of the so called Form C using the web-based tool FORCE available in the EC PP. In the Form C all costs such as Personnel and Travel Costs have to be declared according to the guidelines of the MT, which will be provided as soon as the tool for financial reporting will be available for ARICE in the EC PP. Each beneficiary has to complete a first draft Form C within 30 days after the end of the reporting period. This draft will be evaluated by the MT and if needed, suggestions for corrections will be given to each beneficiary. Only once the MT approves of the Form C the beneficiary has to submit the Form C to their respective FSIGN (Financial Signatory), who will electronically sign and submit the Form C to the PC. Once the Form Cs of all beneficiaries are submitted to the PC, but no later than 60 days after the end of every project period, the PC will submit all Form Cs to the EC. In the frame of the M36, and M48 cost statement, beneficiaries will also have the possibility to adjust the costs declared in previous periods, i.e. at M18 or M36. Adjustments will be made in dedicated forms (called Adjustment Form C) and specific guidance will be provided by MT at the respective reporting period. 5.2.3.2. Process for cost statement preparation and submission to the EC The following process is prepared in order to ensure that all individual cost statements will be submitted to the EC by the Coordinator within 60 days after the end of the reporting period: 1. One month before the end of the reporting period, the MT sends an email to all beneficiaries to inform them about the upcoming financial reporting, and necessary guidance documents, and to remind of the main steps of the process for the costs statements preparation and submission; 2. During the first month after the end of the period, each beneficiary fills in the Draft Form C using the EC PP and informs the MT once the Draft Form C is completed. The Draft Form C, with the explanation on the use of resources, will be validated by the MT who will give suggestions for correction if needed; 3. One month after the end of period, providing that the Form C and the use of resources have been validated by the MT, the beneficiary is allowed to transmit the Form C for electronic signature to its FSIGN; 4. At the latest 45 days after the end of the period, the FSIGN signs the Form C and submits it to the Coordinator; 5. At the latest 60 days after the end of the period, the MT submits all Form C to the EC. 5.3. Certificate of Financial Statements (CFS) Certificate of Financial Statements (CFS) are forms filled in by an external auditor selected by the beneficiary. They contain a number of questions (controls) which the auditor is asked to © ARICE Consortium Page 30 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 answer (check) in verifying the beneficiary accounting and control system or document in relation to the execution of the project. − CFS are only required when the cumulated EU requested financial contribution exceeds 325,000.00 € at the end of the project with the final financial report; − CFS must certify all eligible costs of all periods; − CFS must be submitted according to the templates provided in Annex 5 of the GrA; − CFS are prepared and certified by independent auditors. Competent public officers could also issue them for public bodies and research organizations. − CFS has to be scanned by the beneficiary and uploaded in the Form C. They are submitted to the Coordinator and later the EC as Annex to the Form C solely electronically − The original CFS has to be kept by each beneficiary. No original has to be sent to the Coordinator or the EC. 5.4. Final report on the distribution of the EU financial contribution The final report on the distribution of the EU financial contribution is a table giving the EU contribution finally allocated to each project beneficiary. This table is filled in by the MT after approval of the EU financial contribution distribution by the project‘s beneficiaries, and submitted to the EC via the EC portal. © ARICE Consortium Page 31 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 Deliverable No.8.4 Quality assurance plan ANNEX ANNEX I Contact details for PC, MT, WP leaders, SB and EEAB I.1. Project Coordinator (PC) and Management Support Team (MT) Beneficiary Name Position Email Address AWI Nicole Biebow Coordinator Nicole.Biebow@awi.de AWI Veronica Willmott Project Manager Veronica.Willmott@awi.de Svenja Hildebrandt Programme Manager Svenja.Hildebrandt@awi.de I.2 Work Package Leaders Beneficiary Name Position Email Address SPRS Nicole Biebow WP1 Lead Nicole.Biebow@awi.de WOC Christine Valentin WP2 Lead christine.valentin@oceancouncil.org AWI (APECS) Gerlis Fugmann WP3 Lead Gerlis.Fugmann@awi.de AWI Veronica Willmott WP4 Lead Veronica.willmott@awi.de CSIC Miguel Angel Ojeda WP5 Lead maojeda@utm.csic.es FMI Annu Oikkonen WP6 Lead annu.oikkonen@fmi.fi Arctic Portal Haldór Johanssonn WP7 Lead halldor@arcticportal.org AWI Nicole Biebow WP8 Lead Nicole.Biebow@awi.de AWI Nicole Biebow WP9 Lead Nicole.Biebow@awi.de I.3 Steering Board The ARICE Steering Board is comprised by the MT and the WP Leaders. © ARICE Consortium Page 32 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 ANNEX II Templates for communication material II.1. An official paper letter (Word format) © ARICE Consortium Page 33 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 II.2. A template for publications (Word format) Publication Type of publication Title of publication (in English) Name of the Journal or name of the conference Planned date of publication Other relevant information Abstract Key words Author(s) Full Text © ARICE Consortium Page 34 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 II.3. A template for dissemination activities (Word format) Dissemination activity Type of activity Name of the event (in English) Date Venue Link to event website if available Relevance/objective of the participation in scope of the ARICE project ARICE Participants at the event and their role ARICE materials displayed/distributed Images (Annex images and include credit information) Other relevant information © ARICE Consortium Page 35 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 II.4. A template for relevant news (Word format) ARICE News Title News text Source Website of source OR annex PDF in case it is a printed source II.5. A template for news on a Deliverable (Word format) ARICE News on a Deliverable Title of Deliverable Dissemination level as per DoW (Consortium only, Public or Restricted to a group) Executive summary of the deliverable Responsible institution Deliverable (Annex the PDF) Images/Graphics (Annex images and include credit information) © ARICE Consortium Page 36 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 II.6. Presentation Template (Power Point format) Template for widescreen (16:9) © ARICE Consortium Page 37 of 43 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 II.7. Meeting Agenda (Word format) II.8. Meeting Minutes (Word format) © ARICE Consortium Page 38 of 43 21/01/2019 ARICE – GA No. 730965 © ARICE Consortium Page 39 of 43 Deliverable D8.4 21/01/2019 ARICE – GA No. 730965 Deliverable D8.4 II.9. Meeting Report (Word format) Meeting Report Rapporteur: Meeting details: Name of the conference / workshop Date and location ARICE participant(s) Attendees Please attach list of participants Agenda Please attach agenda Meeting objectives Role of ARICE Meeting outcome / comments Action Items PLEASE ATTACH MEETING MINUTES IF AVAILABLE © ARICE Consortium Page 40 of 43 21/01/2019 ARICE – GA No. 730965 II.10. Deliverable D8.4 Deliverable Template (Word format) Submission of Deliverable Work Package WP 1 Deliverable no. & title D1.1 Title Version Final Creation Date 31.03.2018 Last change 15.04.2018 Status Draft WP lead accepted Executive Board accepted Dissemination level PU-Public PP- Restricted to programme partners RE- Restricted to a group specified by the consortium CO- Confidential, only for members of the consortium Lead Beneficiary AWI (partner 1) Contributors Due date 30.04.2018 Delivery date © ARICE Consortium Page 41 of 43 21/01/2019 ARICE – GA No. 730965 II.11. Deliverable D8.4 Milestone Template (Word format) Submission of Milestone Work Package Milestone no. & title Version Creation Date Last change Status Draft WP lead accepted Executive Board accepted Dissemination level PU-Public PP- Restricted to programme partners RE- Restricted to a group specified by the consortium CO- Confidential, only for members of the consortium Lead Beneficiary Contributors AWI (partner 1) (change accordingly) 1 – AWI, 2 – SPRS, 5 – UAF/CFOS, 9 - WOC, 6 – AP, 10 – IOPAN, 13 – NERC-BAS, 3 - NPI, 4 - ULAVAL, 7 – CSIC-UTM, 11 – FMI, 8 – CNR, 12 - CNRS, 14 – DTU-AQUA Due date Delivery date ARICE © ARICE Consortium Page 42 of 43 21/01/2019 ARICE – GA No. 730965 ANNEX III Deliverable D8.4 Naming conventions Deliverables, Milestones and Internal reports Document File name status Draft/Final ARICE–WP<wp number>-<Del. or Mil. or Rep. Number>-<DDMMYY>V<V_Num> e.g : ARICE-WP1-D1.1-310815-V1 Slideshows/Presentations Document File name status Draft/Final ARICE -<pres title>-<beneficiary>-<DDMMYY>-V<V_Num> e.g : ARICE-This is the title of the presentation-AWI-120615-V1 Meeting agenda, minutes and actions list Document File name status Draft/Final ARICE -WP<wp number>-<Type>-<Date of the meeting>- <Ag. or Min. or List><DDMMYY>-V<V_Num> e.g : ARICE-WP1-EB1-190815-Minutes-190815-V1 Other documents Document File name status Draft/Final ARICE -<doc title>-<beneficiary>-<DDMMYY>-V<V_Num> © ARICE Consortium Page 43 of 43 21/01/2019 

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International Letters of Chemistry, Physics and Astronomy ISSN: 2299-3843, Vol. 37, pp 16-30 doi:10.56431/p-n69wq6 CC BY 4.0. Published by Academic Open Access LTD, 2014 Online: 2014-08-06 Operationally complete work done suggests presence of extra potentials corresponding to repulsive forces Jakub Czajko Science/Mathematics Education Department, Southern University and A & M College, Baton Rouge, LA 70896, USA E-mail address: sunswing77@netscape.net ABSTRACT Operationally complete representation of work done and the corresponding to it potential energy within the usual radial/center-bound, nonrotating gravitational force fields comprises two extra terms: linear nonradial and angular nonradial, in addition to the usual radial term. Since these nonradial terms have negative signs, they suggest presence of potentials corresponding to repulsive forces generated by the very same, usual radial attractive force field. The extra linear nonradial term depends on exposure of an orbiting satellite to the distribution of mass within the field, whereas the extra angular nonradial term also depends on that as well as on exposure of the satellite to density of matter of the mass source that generates the usual, locally dominant radial/center-bound attractive gravitational force field. Keywords: Nonradial potential energy; work done; repulsive potential of gravity 1. INTRODUCTION Stability of macroscopic solids can indicate [the necessity of] presence of some repulsive gravitational forces [1]. If so then perhaps we missed something (inherent to force fields) which warrants the fleeting stability that is visible in nature, especially on macro scale. I will show below that definitely repulsive potentials emerge in the usual radial/center-bound gravitational force field as a necessary mathematical consequence of the field‟s abstract geometric structure that is imparted by interacting potentials of both masses. Since abstract potentials correspond to potential energy spent on the work done by the given force field on a satellite orbiting the field (or on a body traversing it), the geometric structure is of both physical and mathematical provenience tied together. Lobachevski already emphasized that [2]. In 1898 AD Schuster has conjectured that chemical elements with negative masses should be repelled by [the usual radial central] gravitational force field [3]. Nevertheless, I This paper is an open access paper published under the terms and conditions of the Creative Commons Attribution license (CC BY) (https://creativecommons.org/licenses/by/4.0) International Letters of Chemistry, Physics and Astronomy Vol. 37 shall show in what follows that interacting radial potentials can also produce additional nonradial potentials with signs opposite to the usual attractive radial potential, which would correspond thus to a repulsive force. Yet the possibility of existence of nonradial potentials was ignored in former physics. Even when decomposed into the three-dimensional (3D) coordinate system (X,Y,Z), the [generic] potential function V(r) of an attractive radial/center-bound force field due to mass sources was considered only in the radial direction [4,5]. And even when it was generalized to n  3 dimensions, the potential was still defined via n iterated integrals of exactly the same, single generic radial potential function [6]. No other than radial potentials were recognized in Newtonian and Einsteinian theories of gravitation. As a matter of fact, Einstein explicitly declined to consider tangential contributions to the radial impact of gravitational field [7]. Contrary to common perception, center-bound force field is not static during interactions with traversing it bodies. It does not remain the same once it has been created even when its source does not alter any of its field-related attributes. Even if the field‟s source remains unchanged, the field‟s interaction/behavior depends also on all the other entities that happen to move through the field, even if their possible contribution to the field is negligibly small. Although as a whole it could theoretically last forever, the radial field is ever rearranging its abstract internal structure. For it is not really the resultant force vector that actually does act on the particle that moves in it, but each of its coordinate subcomponents (if cast in the reference frame of the given particle affected by the central force field) acts quite independently of all its other coordinate subcomponents – compare [8]. Neglecting the inherent dynamics of the radial force field did lead to several misconceptions in the past, one of which was the former denial of existence of nonradial effects of the usual radial force fields [9]. However, actual existence of nonradial effects and thus associated with them nonradial potentials is confirmed in few experiments [10] and is mathematically necessary [11]. Nevertheless, fairly substantial heap of misconceptions, which emerged in the former physics, has its roots in misguided applications of mathematics, regardless of whether it was done by physicists or mathematicians. Thence a (new) synthetic mathematics is being developed. The new mathematics can invent or synthesize new laws by enhancing those already existing ones by taking cues from curious experimental hints [12]. 2. NEW SYNTHETIC MATHEMATICS FOR PHYSICAL SCIENCES Physics could come up with some new laws of nature and even ensure that they conform to experimental data, but to ascertain that the new laws truly reflect defining features of the reality they describe requires also conceptually sound, as well as operationally complete, (new) synthetic mathematics, which is envisioned not only as a tool to confirm existing physics, but as guide toward new ideas too. I have derived (from experimental hints) new law governing nonradial effects of radial fields [10]. The law retrodicted few previously unexplained experimental results [10]. But that law was neither sufficiently detailed nor general enough to retrodict all related effects nor did it include also some other variables that could influence the phenomena ruled by the law yet were absent in that experiment-driven formula. This has eventually been accomplished in [12] after removing few mathematical obstacles that kept the law from being revealed earlier [9], [13]. In the sense the new synthetic mathematics is necessary to enhance laws of physics. 17 18 ILCPA Volume 37 Although quantum mechanics significantly advanced operational methods of mathematics, it has also inherited several old conceptual deficiencies of the latter, stemming from the subservient role (often termed as that of “handmaid”) ascribed to mathematics by physical sciences. But mathematics has significant, though yet untapped, predictive capabilities whose power extends far beyond the imagination of nonmathematicians and of some mathematicians as well. Through the abstract “eye” of mathematics one might fathom unknown yet, higher laws of nature and mathematics and then actually devise them based upon set of proven operational principles of mathematics [12]. Laws of physics/nature belong in mathematics, not physics, which merely interprets them. Interpretation is important, but it may not be able to reveal the extent to which the scope of the law could be stretched. But in order to stay relevant to physical applications and to the reality we live in, mathematics should take into account also the unanticipated experimental hints that suggest existence of previously unforeseen features of the reality. Instead of leading the efforts to attain predictive enhancements of the already known laws in all sciences (itself included), mathematics concentrated on proving that its deductions are consistent with its own (yet not always forward-looking) assumptions about the realities they pertain to. With its focus directed inwards, mathematics virtually lost its natural leadership role among exact sciences, which it enjoyed since Newton‟s invention (and Leibniz‟s “redemption”) of differential calculus. Rather than upgrading its axiomatic bases upon those experimental hints that indicate ever-expanding features of physical reality, mathematics succumbed to the invidious task of restraining enhancements to the reality by tacitly defining away conceptually inconvenient or just difficult to grasp features of the reality. Mathematics has reduced its role to just confirming what physical sciences happen to discover, even if the discovery was only a tip of the proverbial iceberg but the still submersed (hence not yet fully visible/recognized) iceberg turned out to be very different than its tip, at the expense of its integrity. Mathematics often compromised its methods and even defied some of its own proven operational laws, which tendency led to severe scientific misconceptions in the past [9]. Case in point: In 1773 AD Lagrange realized that Newtonian gravitational force F equals to gradient V = - F of the generic scalar potential function V = 1/r that solves Laplace equation [14]. It was like discovery of a raw mathematical “diamond” that was left unpolished. Mathematics not only invented fake theorem that kept the idea from being further investigated, but it even tacitly violated its own proven laws/rules [9] and suppressed its own proven achievements [15] when they disagreed with some man-made mathematical ideas about the reality. The generic potential is evidently identical to normal/radial curvature of free space, however. Realizing this, one might ask: If so then how about torsion that is actually the second curvature (orthogonal to the primary one), which is (according to differential geometry) absolutely necessary to be considered in general [13]? A single solution of a differential equation is not necessarily its most comprehensive solution. Traditional approach to physical problems used mathematics selectively. It just ignored issues considered as conflicting with formerly accepted paradigms. Despite having knowledge (from introductory level calculus courses) that if the generic rate dW of work done W shall be represented by inner product of the vectors of force F and distance r, then according to proven [16] and operationally complete (hence mandatory) product differentiation rule/law the rate ought to be rendered as dW(Fr) = Fdr + rdF rather than simply as dW := Fdr as it used to be defined in both former physics and International Letters of Chemistry, Physics and Astronomy Vol. 37 19 mathematics. Although the clearly incomplete formula is admissible just for a single standalone force whose magnitude does not vary when the magnitude of distance r = |r| is fixed, for radial/center-bound force fields the cut-short formula is inadequate and leads to incorrect predictions [9]. With more than one source of a force field, the effective vector of force can vary even when the distance is fixed. Hence if distinct sources of the field could not be meaningfully amalgamated into as if single-source radial local field, the incomplete (and thus law-defying) traditional formula is wrong. Notice that the right-hand side (RHS) term rdF has evidently nonradial character because the distance r is fixed there. The fixed radial distance r is just a parameter (in the RHS term) determining generic equipotential surface that surrounds the local gravity center of the locally dominant radial/center-bound force field, i.e. the (central) field generated by the main, single (or amalgamated) local mass source alone. I will show in what follows that the neglected nonradial term can change structure of the entire potential function and thereby also our understanding of gravity. One could imagine several directly or indirectly independent variables upon which the varying rate of force function dF() has to depend, one of which should vary along the equipotential surface, in order for the mathematics (to be deployed for handling the varying force) to make also conceptual sense. Before tackling the issue, however, we must clarify the necessary contextual framework. 3. NONRADIAL EFFECTS OF RADIAL/CENTRAL FORCE FIELDS It has been shown that presence of some other than radial (hence nonradial) components of the usual radial/center-bound force fields really is mathematically unavoidable [13]. Therefore from operationally correct definition of work done rate containing nonradial terms, this fairly comprehensive formula emerged [9]: ( (1) ) where the equipotential term in the middle on the RHS of eq. (1) depends on the varying planar spherical angle α of visibility of the path pointed to by the pointing vector r whose value r =| r| is also the distance between the masses M and m. Here cosα = rp/r with rp as the radius of perihelion and F = |F| the magnitude of force. Since the path was assumed as being originally straightlinear before it was twisted by the local force field, its angle of visibility α can be presumed as planar spherical. At the present depth of inquiry, only motion-independent (hence as if static) nonradial effects are taken into account. They happen along equipotential surfaces where the usual radial potential always remains constant by definition. The angular (middle) term with dα that is determined by the angle α in the eq. (1) yields the equipotential function of the work done rate wrQ(Q(λ)) for which the given field‟s potential energy is spent solely along the equipotential surfaces determined by radius r and codetermined by the field‟s density of matter Q [12]: ( ( )) (2) 20 ILCPA Volume 37 which depends on the function Q(λ) measuring exposure of the orbiting or passing by mass m (assumed as being insignificantly small/negligible in comparison to the, assumed as huge, mass M), which traverses the locally dominant gravitational force field, to the average/constant matter density Q of the source mass M. In other words: the (generally double-curvilinear) angular distance variable λ=rθ (representing a certain spherical angle θ) is measured along the equipotential surface that is codetermined by the average density of matter Q, i.e. the specific gravity of the source mass M that generates the locally dominant gravitational field. The exposure Q(λ) (of the mass m traversing the field due to the mass M) to the field‟s density of matter Q depends on the equipotential angular distance λ, but – at the present depth of inquiry – not on a possible evolution in space/time of the matter density function Q() itself. Since in general the equipotential angular distance λ can be curvilinear, the corresponding to it angle θ is thus spheroidal spherical one. The negative function wrQ(Q(λ)) causes energy/frequency decrease [10]. The coefficient k (to be found from experiments) is needed, indeed, to cover possible impact of a few expected, but unknown yet and thus unaccounted for variables; this shall be explained in more detail elsewhere. For the eq. (2) presumed nonrotating fields and masses, and assumed constant G, M, m, Q, and rp, some of which may, nonetheless, vary depending on certain independently varying variables which are not investigated yet at present depth of inquiry into the gravitational phenomena. For the physical reality we live in is not restricted by what our imagination is able to fathom. The eq. (2) can be rewritten in terms having more direct physical relevance ( ( )) ( ) (3) where ʋ is perimeter volume enclosed by the distance rp to the perihelion from the gravity center of the locally dominant force field [17]. These two equations (2) and (3) are conceptually equivalent, but the eq. (3) is cast in the larger context of purely geometric terms (volume and circumference of an idealized approximately circular orbit, or more likely a part of the orbit as it is seen inside the perihelion). Specific gravity Q = qM/qwater is an intensive (hence comparative) measure of density of matter of the source mass M relative to density of water. Therefore the Ʊ=Q/ʋ denotes relative density of matter enclosed within the perimeter volume. The sack Ʊ can be used for comparing interactions when either the mass density of the source mass M changes (as it can do in exploding or collapsing stars, for instance) or if the perimeter delineated by (shifting/moving) perihelion is altered. The work done rate function wrQ(Q(λ)) that has been reparametrized into one depending on few more variables: wQ(r,rp,Ʊ,λ) could also become more detailed function wQ(r,rp,Ʊ,λ,M,m) if these masses would vary too. The subscript Q in wQ means parameter codetermining the function wQ() even if it is implicit or absent there. Symbols in parentheses following function names identify independent variables. Hence in wrQ(Q(λ)) r and Q play only the role of parameters, just as in wrQ , but when Q is considered as a function Q() then – in the latter incarnation – it is also representing the proxy variable Q(λ) that depends on the angular distance λ and signifies exposure (along the angular distance) to the (assumed as average and thus constant) density of matter Q. Note that – at a deeper than this present level of inquiry – the function Q() can exhibit dependence also on other variables. Parameters can be listed among independently varying variables even if they are fixed for the particular context as constants or averages determining the given situation at hand, in International Letters of Chemistry, Physics and Astronomy Vol. 37 21 which case one might assume that they could vary in general (i.e. in certain other contexts/situations). When the variables are tentatively used as fixed parameters, however, they must not be differentiated or integrated within the same conceptual context defining the situation from which they emerged. This demands distinguishing active variables from passive parameters by unambiguous qualifiers, which was not always accentuated in former physics and mathematics. The distinction is conceptually important to avoid performing illegal operations. 4. FORMER FIELD-NOTIONS SHOULD BE QUALIFIED AS RADIAL Let us review and then rectify several inconsistencies in former physics. The scalar magnitude Γ = |Γ| of the radial Newtonian gravitational force field vector Γ as function of the (radial) distance r = |r| between gravity centers of two interacting masses M and m was traditionally defined in physics as ( ) ( ) | ( )| (4) in the usual, attractive radial/center-bound gravitational force field. Evidently the presumed as locally dominant mass M curves the local space towards itself here and that is why – relativistically speaking – the smaller mass m is forced to orbit the locally dominant mass M. The mass M can also appear as if just bending the trajectory of the mass m if the orbital radius tends to infinity, of course. The radial-only (i.e. partial) rate ∂W(r) of the work done by the field on the traversing it test mass m (which could be moving – in general – under influence of both: the locally dominant gravitational force field and some other, distinct fields) can be defined in terms of either the radial force vector Γ(r) or the radial potential energy function U(r), or the (physical) radial potential function V(r) as: ( ) ( )  ( ) ( ) ( ) ( ) (5) where the physical radial potential V(r) function of the field due to mass M is ( ) ( ) ( ) ( ) (6) where κ = 1/r is generic curvature. The radial potential energy U(r) of the force field relates to the radial physical potential V(r) (as opposed to generic potential which is identical to curvature of the orbit or path around the mass M) in a very similar manner as linear momentum vector p = mv is related to the velocity vector v of the orbiting (or just passing by) test mass m. All these former notions W(r), Γ(r), U(r), V(r) make perfect sense, but only when they are qualified as radial. These field-based notions could be confusing, however, if left unqualified. Examples of such a confusion will be shown below. The qualifier „radial‟ suggests, however, that perhaps some other than radial (i.e. nonradial) field-based notions may also exist beside the radial ones. The sad fact that former physics ignored them did not make the nonradial effects extinct. 22 ILCPA Volume 37 Yet another indicator of necessary presence of certain definitely nonradial notions would be inconsistency of purely radial notions if used alone in general situations of any motions within the radial/center-bound force fields. Although rarely mentioned, this is indeed the case for arbitrary motions within such fields. If magnitude F = |F| of an arbitrary (hence also standalone) force F defined in dynamical terms can be written as generic function of an arbitrary motion (which is not necessarily driven by the locally dominant radial/center-bound gravitational force field alone, but may be influenced also by some other fields) was given by ( ) ( ) ( ) (7) then the arbitrary force F(r) is not really equal to the radial force of gravitation Γ(r) unless a = -ar , i.e. unless the arbitrary acceleration „a‟ is radial, which only happens when all forces can be amalgamated into as if single force field. Hence ( ) ( ) (8) which shows that former balancing of forces was not always correct. If qualified, the eq. (8) indicates need to take also other than radial forces to balance arbitrary forces. Former physics ignored this inadequacy. The disambiguation via explicit qualifiers provides further evidence for necessity of certain nonradial effects [11]. Engines of a spacecraft or laser pulses might create extra contributions to the effective local field, in which case the resulting field cannot be described in radial terms alone. And so does a distant star, even though its impact is detectable only through the rays it emitted, for it is most likely too far away from the local field. Integrating the above differentials gives equivalence of kinetic energy K and potential energy U, but only under the (usually unspoken) assumption that all the motions in the radial/center-bound force field are radial. Without accepting this assumption, equivalence of arbitrary kinetic and radial potential energy is invalid, and thus the alleged consistency of the so-defined energies can fall apart because motions driven by two distinct sources need not always be radial/center-bound: ( ) ( ) ( ) (9) where the radial potential energy U(r) equals only to radial kinetic energy K(r) but these two are not equal to arbitrary kinetic energy K(x) which needs not be radial in general (over a distance x determined by a pointing vector x, which is not always radial). It is known that magnitude/intensity of a field at a point does not determine completely the field at that point [18]. Hence kinetic energy of a mass m driven by more than one source of the effective field may not always be radial: K(x) K(r) = U(r). Mercier already realized that there must be some mixed terms of potential energy [19], [20], even though he has not identified them exactly. If despite the inequality of arbitrary kinetic energy K(x) and radial potential energy U(r) energy should be conserved, an extra energy must exist in order to make/fill up the gap. Chow has pointed out that velocity-dependent interaction is noncentral and thus clearly nonradial [21]. So the possibility of existence of other than radial effects was always present, but it was International Letters of Chemistry, Physics and Astronomy Vol. 37 23 routinely ignored [22]. Although one can always drop some negligibly small nonradial terms in an approximation, if a formula is supposed to be conceptually correct then it must be complete. Even when velocity of the test body that crosses the local force field always remain constant, certain clearly nonradial effects can occur anyway. This is fairly well visible when a nominally massless particle like photon travels at the speed of light and tangentially passes near star such as our Sun, or when some radio waves passed along practically equipotential surface of the Earth [10]. Although the changing distance (to our Sun) from the rays (coming to Earth from distant stars) that pass near our Sun is radial, their trajectory path x along which energy is exchanged (between the rays and the Sun‟s force field) via work done by the Sun‟s field is not exclusively radial. Their path can have both radial components and also components that are not radial with respect to the Sun [10]. The incompletely defined work done could be consistent only if all the force field vectors would always be parallel (which in center-bound fields is impossible by definition), or if the force field vectors could not be resolved into parts parallel and perpendicular to the path of the moving object, but it is always possible by Hamilton‟s theorem [23]. Radial-only notions cannot thus guarantee consistency. I have showed that a certain nonradial work done (and the corresponding to it nonradial potential energy) as well as an extra nonradial force produced by the work/energy emerges from other than radial interactions within the usual central radial/center-bound force field. We have also seen that former radial-only notions cannot account for all field interactions. Also some unanticipated experimental results strongly indicated the presence of definitely nonradial effects of the usual radial gravitational force field, which depend not only on the local field‟s (mass source‟s) density of mass, but inversely also on (an exposure of the orbiting satellite/body to) the local field‟s density of matter [9,10,17]. 5. COMPLETE WORK DONE AND TOTAL POTENTIAL ENERGY At the present depth of inquiry into gravitational phenomena happening in a locally dominant radial/center-bound gravitational force field I am assuming (for the sake of simplicity of this presentation) that all masses of interacting bodies remain constant during their gravitational interaction. This latter assumption means that the gravitational force depends primarily only on the radial distance from the gravity center of the given local force field. Henceforth the term „linear‟, when used in the phrase „nonradial linear‟, can mean „straightlinear‟ (as opposed to „equipotential angular‟, which is bound to an equipotential surface) as well as „curvilinear‟, if the latter is not surface-bound. Since ( ( ) ) ( ( ) we get from the eqs. (1) and ) (4) (10) which is the usual, radial linear part of total work done rate [9]. After integration we obtain two respective amounts of the (radial linear) part of the total work done 24 ILCPA Volume 37 ( ) ( ) (11) where the radial potential that corresponds to the radial part of the work done rises from r to infinity [12]. Notice that the perihelion radius is always smaller than the varying radius/distance (rp ≤ r) by definition and is assumed here as being constant. Similarly also the nonradial linear work done rate is given by the expression: ( ) ( ) ( ) (12) and its integration gives us two amounts of the nonradial linear part of work done ( ) ( ) (13) which complements the radial work done. Notice that while the radius r grows to infinity the magnitude F of the force diminishes, but the minus sign reverses it. It has also been shown that amount wrQ(λ) of nonradial angular work done is ( ) (14) where ʋ is the perimeter volume enclosed by the distance rp to perihelion from the local gravity center of the mass M [12], with the angular distance λ = rθ runs along equipotential surface codetermined by the radial distance r and spherical angle θ. Since specific gravity Q = QM/Qwater is conventionally defined as an intensive (i.e. comparative) measure of density of matter of the source mass M relative to density of water under normal conditions, the parameter Ʊ = Q/ʋ yields the unit of relative density of matter enclosed inside the perimeter volume ʋ = 4π(rp)3/3 [17]. The coefficient k = 1 for perihelion close to surface of the source mass M [12,17]. I have successfully reconciled experiments with waves passing near Sun and Earth using the value k = 1 [10], but k should be determined from experiments for, in general, it could depend on perihelion and perhaps on a few other factors too. As in eq. (1) we got three distinct ) in (11), the nonradial linear term ( components: the usual radial linear term ( ) in (13) and the nonradial angular term wrQ(λ) in (14). Taken together they give us the total amount of work done (within nonrotating radial/center-bound force fields), which – due to energy conservation law – must be equal to the total amount of potential energy U that compensates the work done by the local force field on the gravitational interaction ( ( ( ))) ( ) ( ) ( ( )) (15) which can be written in terms of perimeter volume ʋ and the circumference ц of the perihelion circle and a relative density of matter Ʊ enclosed by the perihelion ( ) (16) International Letters of Chemistry, Physics and Astronomy Vol. 37 where the 2nd nonradial linear term depends on the perihelion radius rp of the mass m orbiting the source mass M of the field, as does the 3rd purely angular nonradial term, which also depends on exposure of the mass m to density of matter Q of the mass source M of the locally dominant field via the angular/equipotential distance λ that is measured along the equipotential parts of a trajectory of the mass m. The perihelion circle is not meant to suggest circular trajectory. It only alludes to the amount of matter enclosed within the volume determined by the perihelion radius. Since the newly emerged measure, namely volume per circumference ᶌ = ʋ/ц is purely structural geometric quantity, which seems to play very important role in linear nonradial potentials, I shall call it henceforth volpercirc, whereas its inverse (circpervol 1/ ᶌ = ц/ʋ) clearly plays defining role for angular nonradial potentials. The total amount of work done and thus consequently also the corresponding to it potential energy of the locally dominant radial/center-bound gravitational force field indicates presence of two relatively small gravitational repulsion terms generated by the usual attractive radial field, due to these two negative nonradial components (linear and angular, respectively) whose signs are opposite to the usual radial potential that generates attractive force between the masses M and m. If the test mass m would orbit the (assumed as huge) mass M on a circle r = rp ( ) ( ) (17) then potential energy U of such an idealized system would increase hyperbolically with radial distance. With increasing radial distance from gravity center, the radial potentials surpass the nonradial ones, which are significant only near surface of the mass M that generates the locally dominant gravitational field – see Fig. 1. Fig. 1. Simplified plot of cross section of the hyperbola‟s right-branch – eq. (17). 25 26 ILCPA Volume 37 Notice that we are talking here about the total potential energy involved in gravitational interactions, not about potential energy of the whole field, which is just one (even though certainly the most important one) of several factors directly relevant to interactions happening within the given gravitational field. Yet because presence of nonradial twist (and thus some θ) is mathematically unavoidable [11], effects of nonradial potential energy of the field‟s interactions cannot be ignored. For mass-sources of the local field with relatively high density of matter the rapidly increasing negative hyperbolic cross-sections on the RHS of the eq. (17) evidently flatten faster with increasing radial distance – see Fig. 1. The plot in Fig. 1 shows total potential energy (y) of interactions increasing with the radial distance r = x for Earth (blue) with Q  5.52 and Saturn (red) with Q  0.687. This plot represents the eq. (17) approximated for these two planets. Yet if the eq. (17) must retrodict the radial Newtonian gravity, the condition (18) is necessary, but only as a limit. It is up to experiments to determine the k, which could be a function of several other factors. Since the (minimal) angle θ (or the induced nonradial twist as it might be called) could vary even when the average density of matter Q remains constant, therefore – at the present depth of inquiry –the coefficient k may vary too. This seemingly confirms the fact that there can be no absolutely empty physical space and no infinitely small singularity possible. Two unbiased experiments have been reconciled with k  1 with nonradial effects happening fairly close to surfaces of the Sun and Earth [10]. If the angle θ is tied to the density of matter Q of the mass source of the local force field, the field should exhibit an intrinsic rotation, which is neglected for the time being. These tentative conclusions agree with realistic interpretation of infinity [24], [25]. These and other related issues shall be discussed in more detail elsewhere. 6. NONRADIAL POTENTIALS COMPLEMENT THE RADIAL ONE While Newton, Einstein and most subsequent investigators of gravitational phenomena usually considered the proverbial gravitating apples as being only under the influence of single-source (locally dominant or perhaps amalgamated) gravitational field and therefore as freely falling directly towards the local gravity center, the depth of their inquiry was always restricted to radial-only effects of gravitation. For Newton any exceptions from purely radial (hence center-bound) trajectory were essentially of procedural nature, i.e. caused only by a preexisting momentum of the locally bound satellite or a body passing by. Due to lack of the notion of potential of gravitational field at the time when Newton came up with his theory of gravitation he did not see his force as arising from clash of the field‟s potentials. His greatest achievement was representing changes to physical concepts by differentials. But since Lagrange and especially after Frenet, the unavoidable nonradial potentials should not be ignored [11]. Besides, both Newton and Einstein felt justified in their rejection of possible other than radial effects of the usual radial/center-bound gravitational fields by the Galilei‟s misguided conclusion that constitution of matter (and in particular thus density of matter) does not affect International Letters of Chemistry, Physics and Astronomy Vol. 37 27 gravitational phenomena, which he had drawn from his too oversimplified experiments with dropping objects from the leaning tower of Pisa. Even though the simplistic Galilei‟s conclusion was instrumental for both Newton and Einstein, it is nevertheless mathematically unacceptable [17] and I have shown in [10] that two ingenious modern experiments defied it [17]. We should certainly applaud Galilei for easing the way for Newton to come up with his relatively simple equation for radial gravitational interactions, but we should not keep on fooling ourselves by perpetuating his mathematically deficient reasoning, which was step forward back then but is hampering physics today. For Einstein the exceptions were due to curvature of the spacetime – hence of a structural nature. However, two distinct objects at the same point in space could have very different trajectories, depending on their preexisting momentum, which means that each object carries its own peculiar spacetime as if on its back, which fact makes them incomparable – see [26,27]. Hence the structure of Einsteinian curved spacetime seems to be flexible, as if personalized spatial structure, neither a solid nor a geometric space. Therefore its potential energy cannot be localized. Since the extra nonradial effects have emerged from operationally complete representation of potential energy, they indeed complement the general theory of relativity (GTR) which had been deliberately restricted by Einstein to only radial gravitational interactions [7]. Besides, GTR and GTR-like theories of gravitation published prior to year 2000 AD (i.e. before the theory of nonradial gravitational effects was envisioned in [28] and then invented in [10]), merely decomposed the magnitude of the, essentially still radial, effect of the curved spacetime (or quasi-Newtonian radial force) into the global 3D coordinates tied to local gravity center of the primary mass source that generates the locally dominant gravitational field. The GTR‟s primary equations of purely radial gravitational interactions did not take into account any physical magnitudes other than those already present in the Newtonian theory, but they changed the way the radial gravitational potentials are being distributed in spacetime. All former theories of gravitation assumed that interactions within gravitational fields were determined by density of mass alone, based on the Galilei‟s erroneous conclusion that density of matter does not matter. The eq. (17), however, also shows how potentials involved in gravitational interactions are being composed (as opposed to just decomposition of radial force or radial potential onto a three-dimensional (3D) coordinate system, which still does not turn the decomposition into a composition). The distinction between composing versus decomposing vectors is very important. Riemann has already pointed out that even when decomposed into 3D coordinate system (X,Y,Z), the [generic] potential function V(r) of attractive radial/center-bound force field due to mass sources was still considered only in the radial direction alone [4,5]. If force is simplistically viewed as just a passive property of radial/center-bound force field, then it is radial. But the active force involved in interactions is being composed from potentials of the interacting bodies – which fact we learned from Aharonov-Bohm effect – [17], and such active force is not always radial [11]. Decomposition is merely redistribution or deconstruction of the usual radial-only potentials – it is just a projection of the radial potentials on the coordinate axes. Composition, however, shows where their subcomponents originated from. New subcomponents could emerge in composing potentials when more defining factors are taken into consideration. Decomposing radial-only potentials cannot add any new predictors. To insist that any derived equation is final, is an insult to one‟s own and the readers‟ intelligence. At a certain deeper 28 ILCPA Volume 37 than the present level of inquiry one can find that Sun‟s gravity depends also – to some degree – on its average surface temperature, for instance. More precise equation can reveal tiny dependence of gravity on the abundance of its elements, hence on the Sun‟s age. Moreover, at the present depth of inquiry into gravitational interactions the eq. (17) shows the impact of exposure of the orbiting (or just passing-by) mass m to (average) composition/density of matter of the field‟s source mass M (though not to the internal structural composition of particulate matter itself yet). In the sense the nonradial theory complements radial theories of gravitation without defying their tenets. The distribution of purely radial interactions in the GTR is not questioned. All former theories of gravitation were virtually (though not always openly) challenged by the mathematical incompleteness of the faulty previous definition of potential energy and work done, which they inherited from ingenious work of Lagrange [9], likely due to his overly reliance on the aforesaid oversimplified Galilei‟s conclusion, I presume. No theory can tacitly violate select (because they seem inconvenient and/or their consequences are difficult to grasp) mandatory operational laws/rules of mathematics and remain unscathed. Former incomplete mathematics resulted not only in misguided theories of physics, but it also adversely affected other related reasonings and their applications to other mathematical theories. With deepening levels of inquiry into gravitational phenomena our previous laws of physics will grow more detailed. The old, coarse laws will be enhanced by effects revealed in new experiments, which should not be ignored in mathematics if it wants to stay relevant to reality in general and physical sciences in particular. 7. CONCLUSIONS Total potential energy of gravitational field spent on work done by the radial field of gravity depends on perihelion radius/distance as well as on the curvilinear distance traveled along equipotential surfaces and also (inversely though) on the density of matter of the source mass that generates the locally dominant field. New law for gravitational interactions in terms of work done and potential energy has been synthesized on the basis of operationally complete mathematical representation of the work done rate and upon its relation to potential energy. The new law suggests that, in addition to the usual radial gravitational potential, two extra repulsive potentials arise: one is linear nonradial and the other is angular nonradial. The extra potentials modify the former, usual radial potential function. The operationally complete (and thus mathematically legitimate) potential energy U(r,λ) varies not only as a result of position vector changing within the usual locally dominant radial/center-bound gravitational force field, but it is also codetermined by several other variables and fixed parameters, one of which is (angular/equipotential exposure to) average density of matter of the local field. References [1] Jeffreys H., Jeffreys B., Methods of mathematical physics. Cambridge: Cambridge Univ. Press, 2001, p.202. International Letters of Chemistry, Physics and Astronomy Vol. 37 [2] Licis N.A., Philosophical and scientific meaning of ideas of N.I. Lobachevskii. Riga, 1976, pp. 58, 372 [in Russian]. [3] Hughes R.J., Contemp. Phys. 1993 34 177-91. [4] Riemann B., Schwere, Elektricitӓt und Magnetismus. Hannover: Carl Rümpler, 1876, p. 9ff. [5] Gutzwiller M.C., Chaos in classical and quantum mechanics. New York: Springer, 1990, p. 101. [6] Kasner E., De Cicco J., PNAS USA 38 (1952) 145-148. [7] Einstein A., The Foundations of the General Theory of Relativity. [pp.111-164 in: H.A. Lorentz et al. The principle of relativity. Dover, New York 1923, see p.161]. [8] Sokolnikoff I.S., Sokolnikoff E.S., Higher mathematics for engineers and physicists. New York: McGraw-Hill, 1941, p. 218. [9] Czajko J., Stud. Math. Sci. 7(2) (2013) 25-39. [10] Czajko J., Chaos, Solit. Fract. 11 (2000) 2001-2016. [11] Czajko J., Appl. Phys. Res. 3(1) (2011) 2-7. [12] Czajko J, Stud. Math. Sci. 7(2) (2013) 40-54. [13] Czajko J., Chaos Solit. Fract. 20 (2004) 683-700. [14] Birkhoff G. (Ed.) A source book in classical analysis. Cambridge, MA: Harvard Univ. Press, 1973, pp.335, 360. [15] Czajko J., International Letters of Chemistry, Physics and Astronomy 17(2) (2014) 220-235. [16] Bers L., Calculus I. New York: Holt, Rinehart and Winston, 1967, p. 216f. [17] Czajko J., International Letters of Chemistry, Physics and Astronomy 11(2) (2014) 89-105. [18] Rylov Yu.A., Sov. Phys. Dokl. 7(6) (1962) 536-538. [19] Mercier A., Analytical and canonical formalism in physics. Amsterdam: North-Holland, 1959, p. 122. [20] Mercier A., Speculative remarks on physics in general and relativity in particular. [pp.295-303 in: De Sabbata, V. & Weber, J. (Eds.) Topics in theoretical and experimental gravitation physics. London: Plenum Press, 1977]. [21] Chow T.L., Classical Mechanics. New York: Wiley, 1995, p. 35. [22] Goldstein H., Poole C., Safko J., Classical mechanics. San Francisco: Addison-Wesley, 2002, p. 4ff. [23] Doran C., Lasenby A., Gul S., Found. Phys. 23(9) (1993) 1175-1201, see p. 1186. [24] Czajko J., Chaos, Solit. Fract. 21 (2004) 261-271. [25] Czajko J., Chaos, Solit. Fract. 21 (2004) 501-512. [26] Geroch R., General relativity from A to B. Chicago: The Univ. of Chicago Press, 1978, pp. 166, 171. 29 30 ILCPA Volume 37 [27] O‟Neill B., Semi-Riemannian geometry with applications to relativity. New York: Academic Press, 1983, p. 171. [28] Czajko J., Chaos, Solit. Fract. 11 (2000) 1983-1992. ( Received 12 July 2014; accepted 22 July 2014 ) 

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Atmos. Chem. Phys., 21, 11759–11779, 2021 https://doi.org/10.5194/acp-21-11759-2021 © Author(s) 2021. This work is distributed under the Creative Commons Attribution 4.0 License. The reduction in C2H6 from 2015 to 2020 over Hefei, eastern China, points to air quality improvement in China Youwen Sun1,2 , Hao Yin1,2 , Cheng Liu1,2,3 , Emmanuel Mahieu4 , Justus Notholt5 , Yao Té6 , Xiao Lu7 , Mathias Palm5 , Wei Wang1 , Changgong Shan1 , Qihou Hu1 , Min Qin1 , Yuan Tian8 , and Bo Zheng9 1 Key Laboratory of Environmental Optics and Technology, Anhui Institute of Optics and Fine Mechanics, HFIPS, Chinese Academy of Sciences, Hefei 230031, China 2 Key Laboratory of Precision Scientific Instrumentation of Anhui Higher Education Institutes, University of Science and Technology of China, Hefei 230026, China 3 Anhui Province Key Laboratory of Polar Environment and Global Change, USTC, Hefei 230026, China 4 Institute of Astrophysics and Geophysics, University of Liège, 4000 Liège, Belgium 5 Institute of Environmental Physics, University of Bremen, 28334 Bremen, Germany 6 Laboratoire d’Etudes du Rayonnement et de la Matière en Astrophysique et Atmosphères (LERMA-IPSL), Sorbonne Université, CNRS, Observatoire de Paris, PSL Université, 75005 Paris, France 7 School of Atmospheric Sciences, Sun Yat-sen University, Zhuhai 519082, China 8 Institute of Physical Science and Information Technology, Anhui University, Hefei 230601, China 9 Institute of Environment and Ecology, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, China Correspondence: Cheng Liu (chliu81@ustc.edu.cn) and Hao Yin (yhyh95@mail.ustc.edu.cn) Received: 9 January 2021 – Discussion started: 22 March 2021 Revised: 1 June 2021 – Accepted: 13 July 2021 – Published: 6 August 2021 Abstract. Ethane (C2 H6 ) is an important greenhouse gas and plays a significant role in tropospheric chemistry and climate change. This study first presents and then quantifies the variability, sources, and transport of C2 H6 over densely populated and highly industrialized eastern China using groundbased high-resolution Fourier transform infrared (FTIR) remote sensing along with atmospheric modeling techniques. We obtained a retrieval error of 6.21 ± 1.2 (1σ )% and degrees of freedom (DOFS) of 1.47 ± 0.2 (1σ ) in the retrieval of C2 H6 tropospheric column-averaged dry-air mole fraction (troDMF) over Hefei, eastern China (32◦ N, 117◦ E; 30 m a.s.l.). The observed C2 H6 troDMF reached a minimum monthly mean value of 0.36 ± 0.26 ppbv in July and a maximum monthly mean value of 1.76 ± 0.35 ppbv in December, and showed a negative change rate of −2.60 ± 1.34 % yr−1 from 2015 to 2020. The dependencies of C2 H6 troDMF on meteorological and emission factors were analyzed using generalized additive models (GAMs). Generally, both meteorological and emission factors have positive influences on C2 H6 troDMF in the cold season (December–January– February/March–April–May, DJF/MAM) and negative influences on C2 H6 troDMF in the warm season (June–July– August/September–October–November, JJA/SON). GEOSChem chemical model simulation captured the observed C2 H6 troDMF variability and was, thus, used for source attribution. GEOS-Chem model sensitivity simulations concluded that the anthropogenic emissions (fossil fuel plus biofuel emissions) and the natural emissions (biomass burning plus biogenic emissions) accounted for 48.1 % and 39.7 % of C2 H6 troDMF variability over Hefei, respectively. The observed C2 H6 troDMF variability mainly results from the emissions within China (74.1 %), where central, eastern, and northern China dominated the contribution (57.6 %). Seasonal variability in C2 H6 transport inflow and outflow over the observation site is largely related to the midlatitude westerlies and the Asian monsoon system. Reduction in C2 H6 abundance from 2015 to 2020 mainly results from the decrease in local and transported C2 H6 emissions, which points to air quality improvement in China in recent years. Published by Copernicus Publications on behalf of the European Geosciences Union. 11760 1 Y. Sun et al.: Reduction in C2 H6 from 2015 to 2020 over Hefei Introduction Ethane (C2 H6 ) is an important greenhouse gas and one of the most abundant volatile organic compounds (VOCs) in the atmosphere (Abad et al., 2011; Singh et al., 2001; Steinfeld, 1998). Although C2 H6 is much less abundant than methane (CH4 ) and also less efficient relative to mass, it plays a significant role in tropospheric chemistry and climate change (Tzompa-Sosa et al., 2017). In the presence of nitrogen oxides (NOx = NO + NO2 ), C2 H6 oxidation can enhance tropospheric ozone (O3 ) generation, which shows a positive radiative influence on climate (Sun et al., 2018a) and threatens crop yields (Sun et al., 2018a; Van Dingenen et al., 2009) and human health (Sun et al., 2018a; Tzompa-Sosa et al., 2017). In addition, as a major source of acetaldehyde (CH3 CHO), C2 H6 has a great impact on the production of peroxyacetyl nitrate (PAN) which is a key reservoir species of NOx (Fischer et al., 2014). The main sink of tropospheric C2 H6 is predominantly destruction via reaction with the hydroxyl radical (OH) (Xiao et al., 2008), which determines the residence time of most tropospheric species (Steinfeld, 1998). As a result, tropospheric C2 H6 can decrease the atmospheric oxidative capacity and indirectly impact the climate by extending the CH4 lifetime (Monks et al., 2018; Taylor et al., 2020). Atmospheric C2 H6 has a relatively long residence time of a few months (Franco et al., 2016), allowing it to undergo intercontinental transport. As a result, observations of C2 H6 can be assimilated into a chemical transport model to estimate nonlocal emissions and air quality, and provide valuable insights into model biases of C2 H6 simulations (Tzompa-Sosa et al., 2017). On a global scale, the main sources of C2 H6 are leakage from production, processing, and transport of natural gas (62 %), and biofuel combustion (20 %) and biomass burning emission (18 %) largely occurred in the Northern Hemisphere (NH) (Franco et al., 2016; Xiao et al., 2008). Additional minor sources of C2 H6 are from biogenic and oceanic sources. However, on a regional scale, the proportion of each C2 H6 source may show large differences. The natural gas leakage contribution can reach 80 % of C2 H6 emissions in regions with active oil and natural gas production (Gilman et al., 2013), where C2 H6 emissions are highly correlated with CH4 emissions. In such regions, C2 H6 can be applied as a tracer for the separation of fossil fuel CH4 emissions from multiple methane (CH4 ) sources (e.g., oil and gas, cows, wetlands, and rice yield) (McKain et al., 2015; Roscioli et al., 2015). The C2 H6 abundance in the Southern Hemisphere (SH) is much lower than that in the NH, as the anthropogenic C2 H6 sources are low in the SH and the residence time of C2 H6 is shorter than the interhemispheric exchange rate. Many studies have concluded that C2 H6 in the SH is primarily emitted from biomass burning and is closely correlated with CO and HCN emissions (Notholt et al., 2000; Rinsland et al., 2002; Vigouroux et al., 2012; Zeng et al., 2012). Atmos. Chem. Phys., 21, 11759–11779, 2021 C2 H6 is one of the target gases of a global groundbased Fourier transform infrared spectroscopy (FTIR) network, namely the infrared working group (IRWG) of the Network for Detection of Atmospheric Composition Change (NDACC) (De Mazière et al., 2018). FTIR time series of C2 H6 with different time periods have been reported at many stations for the validation of satellite data or chemical model simulation (Abad et al., 2011; Franco et al., 2015, 2016; Glatthor et al., 2009) or for the evaluation of local air quality and air pollutant transport caused by anthropogenic emission and biomass burning (Angelbratt et al., 2011; Lutsch et al., 2016, 2019; Nagahama and Suzuki, 2007; Rinsland et al., 2002; Simpson et al., 2012; Viatte et al., 2015, 2014; Vigouroux et al., 2012; Zeng et al., 2012; Zhao et al., 2002). Several FTIR sites have observed the decrease in C2 H6 over the 1990–2010 period and have characterized consistent interannual trends in the −1 to −2.7 % yr−1 range (Franco et al., 2015, 2016; Simpson et al., 2012; Zeng et al., 2012). This declining trend has been largely attributed to the reduction in global fugitive emissions (Franco et al., 2015; Simpson et al., 2012). Recently, several studies concluded that the long-term decline in C2 H6 in the NH reversed from 2009 onwards (Franco et al., 2015, 2016). Using ground-based FTIR C2 H6 total columns derived at five selected NDACC sites, Franco et al. (2016) characterized the C2 H6 evolution from 2009 to 2015 and determined growth rates of ∼ 3 % yr−1 at remote sites and of ∼ 5 % yr−1 at midlatitudes. This change is mainly attributed to the exploitation of shale gas and tight oil reservoirs in North America (Franco et al., 2016; Helmig et al., 2016). The NDACC network has been operating for almost 3 decades around the globe (De Maziere et al., 2018; Sun et al., 2018a). However, most instruments are located in Europe and Northern America, the number of observation sites in the rest parts of world remains sparse, and there is only one qualified observations site in China, i.e., the Hefei site (32◦ N, 117◦ E; 30 m a.s.l.), located in a densely populated and highly industrialized area in eastern China (Sun et al., 2018a). The Hefei site is not yet affiliated with the NDACC network, but its observation routine has followed the NDACC standard convention since 2015 (Sun et al., 2018a). As the consequence of a series of actions for emission control, air pollution over China in recent years has been significantly decreased (Zhang et al., 2019; Zheng et al., 2018). However, the atmospheric pollution over densely populated and highly industrialized eastern China is still severe (Zhang et al., 2019; Zheng et al., 2018). The complexity, extension, and severity of the atmospheric pollution in eastern China are still unrivaled compared with the rest of world (Lu et al., 2018; Zheng et al., 2018). FTIR observations at Hefei have been used extensively for the evaluation of satellite data (Tian et al., 2018; Wang et al., 2017), chemical model simulation (Tian et al., 2018; Yin et al., 2020, 2019), local air quality (Shan et al., 2019; Sun et al., 2018a), and the transport https://doi.org/10.5194/acp-21-11759-2021 Y. Sun et al.: Reduction in C2 H6 from 2015 to 2020 over Hefei of air pollutants caused by anthropogenic and biomass burning emissions (Sun et al., 2018a, 2000; Y. Sun et al., 2021). In this study, we first present and then quantify the variability, sources, and transport of C2 H6 over densely populated and highly industrialized eastern China using FTIR observation, GEOS-Chem model simulation, and the analysis of the meteorological fields. The seasonality and interannual variability of C2 H6 over Hefei, eastern China, from 2015 to 2020 are investigated. The dependencies of C2 H6 on meteorological and co-emitted gases (hereafter emission factors) are analyzed using generalized additive models (GAMs) (Wood and Simon, 2004). The ground-based FTIR C2 H6 time series are, for the first time, applied to evaluate the GEOS-Chem model with respect to the simulation of C2 H6 for specific polluted regions over eastern China. Furthermore, we run a series of GEOS-Chem sensitivity simulations to quantify the relative contributions of various source categories and regions to the observed C2 H6 variability. The three-dimensional (3D) transport inflow and outflow pathways of C2 H6 over the observation site are finally determined by the GEOS-Chem sensitivity simulations and the analysis of the meteorological fields. This study can not only enhance the understanding of regional emission, transport, and air clean actions over eastern China but can also contribute to form new reliable remote sensing data in this sparsely monitored region for climate change research. The next section describes the retrieval of the FTIR tropospheric column-averaged dry-air mole fraction (troDMF) of C2 H6 , the configuration of GEOS-Chem model simulation, and the GAMs regression approach. Section 3 reports the variability of C2 H6 troDMF and a comparison with the GEOS-Chem simulation. Section 4 reports the GAMs regression results and the interpretation. Section 5 reports the results for source attribution using a GEOS-Chem sensitivity simulation and the analysis of the meteorological fields. We conclude the study in Sect. 6. 2 2.1 Methods and data C2 H6 troDMF retrieval The C2 H6 troDMF time series were calculated using direct solar absorption spectra saved with a FTIR spectrometer in operation at Hefei, eastern China (Sun et al., 2018a; Tian et al., 2018). A site description and instrumentation can be found in Sun et al. (2018a). Briefly, the FTIR observatory includes a high-resolution FTIR spectrometer (IFS125HR, Bruker) and a solar tracker (Solar Tracker A547, Bruker). The instrument has been operating continuously since its installation; however, short data gaps of up to 8 months have occurred due to a scanner problem between November 2016 and July 2017. This FTIR observatory alternately saved near-infrared (NIR) and middle-infrared (MIR) solar spectra in routine observations, with spectral ranges from https://doi.org/10.5194/acp-21-11759-2021 11761 4000 to 11 000 cm−1 and from 500 to 8500 cm−1 , respectively (Tian et al., 2018). The NIR and MIR spectra are saved with different spectral resolutions, but both of them can be used to retrieve total columns and volume mixing ratio (VMR) profiles of a variety of trace gases in the atmosphere. The MIR spectra used in the present work are saved with a spectral resolution of 0.005 cm−1 following the requirements of the NDACC standard convention (http://www.ndacc.org/, last accessed on 27 December 2020). The FTIR instrument is equipped with a KBr beam splitter, a filter centered at 2800 cm−1 , and an InSb detector for C2 H6 measurements. The number of C2 H6 measurements on each measurement day varied from 1 to 17 with an average of 6. In total, there were 743 d of qualified measurements between 2015 and 2020. In this study, the VMR profile of C2 H6 was first retrieved using the SFIT4 algorithm, updated from SFIT2 (Pougatchev et al., 1995), and implementing the optimal estimation method (Rodgers, 2000). The C2 H6 troDMF was then calculated by taking a weighting average of the C2 H6 VMR profile and the air mass using a fixed tropospheric altitude. The C2 H6 VMR profile was retrieved in a broad window of 2976–2978 cm−1 . The VMR profiles of CH4 and H2 O and column of O3 were also retrieved along with the C2 H6 VMR profile for minimizing the atmospheric absorption interference. Spectroscopic absorption parameters of all gases are based on the atm16 line list from the compilation of Geoffrey Toon (Y. Sun et al., 2021). The a priori vertical profiles of temperature, H2 O, and pressure were interpolated from the National Centers for Environmental Protection (NCEP) reanalysis data, and the a priori vertical profiles of other gases were from the statistical averages of the Whole Atmosphere Community Climate Model version 6 (WACCM) simulations from 1980 to 2020. The diagonal elements of the a priori covariance matrices Sa and the measurement noise covariance matrices Sε were set to the standard deviation (SD) of the WACCM simulations and the inverse square of the signalto-noise ratio (SNR) of each spectrum, respectively (Franco et al., 2015). The non-diagonal elements of both Sa and Sε were set to zero. The instrument line shape (ILS) of the FTIR instrument deduced from optical path alignment diagnosis with a low-pressure HBr cell was adopted in the retrieval (Hase, 2012; Sun et al., 2018b). For each retrieval, the averaging kernels reflect the sensitivity of the retrieved profile to the real profile. The area of the averaging kernels at a specific height is calculated as the sum of the elements of the corresponding averaging kernels (Pougatchev et al., 1995). It represents the fraction of the retrieval at that height that comes from the measurement rather than from the a priori information (Rodgers, 2000). A value close to unity at a specific height indicates that the retrieved profile at that height is nearly independent of the a priori profile and is, thus, from the measurement. The trace of the averaging kernel matrix is defined as degrees of freedom for signal (DOFS) and it quantifies the number of indepenAtmos. Chem. Phys., 21, 11759–11779, 2021 11762 Y. Sun et al.: Reduction in C2 H6 from 2015 to 2020 over Hefei Figure 1. (a) Averaging kernels and their area scaled by a factor of 0.1, (b) cumulative sum of degrees of freedom for signal (DOFS), and (c) volume mixing ratio (VMR) profile of a randomly selected C2 H6 retrieval over Hefei, eastern China. dent pieces of information in the retrieved profile. Figure 1 shows the averaging kernels as well as their area, cumulative sum of DOFS, and VMR profile of a randomly selected at Hefei. Ground-based FTIR C2 H6 observations at Hefei have a sensitivity of larger than 0.7 from ground to about 10 km altitude, indicating that the retrievals are mainly sensitive to the troposphere. This also means that more than 70 % of the retrieved profile information below 10 km comes from the measurement, or in other words, that the a priori signal impacts the retrieval by less than 30 % (Fig. 1a). The typical DOFS obtained at Hefei over the total atmosphere for C2 H6 is 1.69 ± 0.29 (1σ ), meaning that we can roughly provide two pieces of information on the vertical profile (Fig. 1b). The shape of the retrieved profile is heavily weighted toward the lower troposphere. As shown in Fig. 1c, the C2 H6 concentration decreased by 72.7 % with an increase in the height from surface to 2 km and kept decreasing slowly in the remaining part of the atmosphere until it approached around zero in the stratosphere and above. The C2 H6 partial column below 10 km accounted for 88.6 % of the C2 H6 total column. This percentage is expected to show less seasonal variation, as the shape of the retrieved profile is similar to the shape of the a priori profile due to the low DOFS (Fig. 1c). As a result, in subsequent analysis, the C2 H6 VMRs averaged between the surface and 10 km are selected as representatives of C2 H6 troDMF. The selected tropospheric layer (from the surface up to 10 km) corresponds to 1.47 ± 0.2 (1σ ) of DOFS and can be used with confidence. This selected layer is totally within the tropopause height (∼ 16 km) at Hefei over four seasons (Sun et al., 2020). The Hefei site is located in the northeastern margin of a GEOS-Chem grid cell (Fig. 2). Atmos. Chem. Phys., 21, 11759–11779, 2021 Table 1. Error budget and degrees of freedom (DOFS) for signal of a randomly selected C2 H6 troDMF retrieval over Hefei, eastern China. Error source Input value Temperature uncertainty Zero-level uncertainty Retrieval parameter uncertainty Measurement error Interfering species uncertainty Smoothing uncertainty 1σ of NCEP 1% ∗ 1/SNR2 SD of WACCM ∗ Error budget 1.69 % 1.45 % < 0.1 % 0.53 % 0.11 % 0.37 % Total random error / 2.32 % Background curvature uncertainty Field-of-view uncertainty Optical path difference uncertainty Solar zenith angle uncertainty Phase uncertainty Instrument line shape (ILS) uncertainty Line temperature broadening uncertainty Line intensity uncertainty Line pressure broadening uncertainty Total systematic error 1% 1% 1% 1% 1% 1% 5% 5% 5% / Total errors / 6.21 % DOFS (–) / 1.47 0.14 % < 0.1 % < 0.1 % < 0.1 % < 0.1 % < 0.1 % 0.4 % 5.12 % 0.93 % 5.48 % ∗ These input values for error budget estimation are based on the retrieval output. This selected layer also ensures that the lines of sight of all observations are totally within the same grid cell. We calculated the error budget for C2 H6 retrieval at Hefei following the formalism of Rodgers (2000) and separated all error components into systematic or random errors according to whether they vary steadily or randomly over consechttps://doi.org/10.5194/acp-21-11759-2021 Y. Sun et al.: Reduction in C2 H6 from 2015 to 2020 over Hefei 11763 Figure 2. Geographical regions used for GEOS-Chem sensitivity simulations. The numbers À–Å represent western, northern, central, eastern, and southern China, and the rest of world, respectively. See Table 3 for latitude and longitude delimitations. Daily mean values of C2 H6 troDMF on 1 January 2017 provided by GEOS-Chem BASE simulation were selected as representative of wintertime enhancement in eastern China. The base map of this figure is created by the Python “Basemap” package. utive measurements. The random, systematic, and combined error budgets for the selected tropospheric layer (from the surface up to 10 km) are summarized in Table 1. The input covariance matrix of temperature is based on the differences between sonde and an ensemble of NCEP temperature profiles near Hefei, leading to 2–5 K in the troposphere and 3–7 K in the stratosphere. For each of the interfering gases, the corresponding covariance matrix is obtained with the WACCM v6 climatology. The input covariance matrix of the measurement error is based on the inverse square of the SNR of each spectrum. We regularly use a low-pressure HBr cell to diagnose the misalignment of the FTIR spectrometer and to realign the instrument when indicated. The FTIR spectrometer at Hefei is assumed to be not far from the ideal condition, and the input uncertainties for zero level, background curvature, field of view, optical path difference, solar zenith angle, interferogram phase, and ILS are estimated to be 1 %. For the C2 H6 spectroscopic absorption coefficients, the line list in atm16 follows HITRAN2012 (Rothman et al., 2013), and we use 5 % for line intensity and pressure- and temperature-broadening coefficients. For the retrieval parameter and smoothing error, the input covariance matrices are prescribed from the optimal estimation retrieval outputs. To estimate the retrieval error of C2 H6 troDMF at Hefei, the elements of all gain matrices were set to zero for the altitudes outside the selected layer. The contributions of all error components to C2 H6 troDMF retrieval at Hefei are summa- https://doi.org/10.5194/acp-21-11759-2021 rized in Table 1. The dominant random errors are from temperature uncertainty (1.69 %) and the zero-level uncertainty (1.45 %), and the dominant systematic error is from the line intensity uncertainty (5.12 %). Total random and systematic errors are estimated to be 2.32 % and 5.48 %, respectively. Total retrieval error, calculated as the square root sum of the squares of total random and systematic errors, is estimated to be 6.21 %. In order to exclude measurements that were seriously affected by instable weather conditions or by the a priori profile due to low measurement information content under less favorable observational conditions (e.g., around noontime when the probed atmosphere is thinner or in summer when C2 H6 is less abundant), the FTIR measurements saved with a solar intensity variation (SIV) of larger than 10 % or retrievals with total DOFS of less than 0.7 or a root mean square (RMS) of fitting residuals of larger than 2 %, which accounted for 11.2 % of total measurements, were excluded from this study. 2.2 GEOS-Chem sensitivity simulation The relative contribution of various source categories and regions to the observed C2 H6 variability were quantified by a series of sensitivity simulations using the GEOS-Chem chemical model version 12.2.1 (Bey et al., 2001) (http:// geos-chem.org, last access on 24 August 2020). All simulations implemented a universal tropospheric–stratospheric Atmos. Chem. Phys., 21, 11759–11779, 2021 11764 Y. Sun et al.: Reduction in C2 H6 from 2015 to 2020 over Hefei Table 2. Anthropogenic C2 H6 emissions in China by region and category for the 2015 and 2019 Multi-resolution Emission Inventory for China (MEIC) emission inventories. WR refers to western China, NR refers to northern China, CR refers to central China, ER refers to eastern China, and SR refers to southern China. Region Industry (Tg yr−1 ) Power plant (Tg yr−1 ) Residential (Tg yr−1 ) Transport (Tg yr−1 ) Sum (Tg yr−1 ) WR 2015 2019 Change 0.084 0.097 15.36 % < 0.01 < 0.01 82.54 % 0.011 0.011 −6.61 % < 0.01 < 0.01 −3.41 % 0.1 0.112 12.12 % NR 2015 2019 Change 0.241 0.241 0.04 % < 0.01 < 0.01 2.51 % 0.125 0.105 −15.96 % 0.026 0.025 −4.38 % 0.394 0.373 −5.32 % CR 2015 2019 Change 0.144 0.15 4.68 % < 0.01 < 0.01 −7.00 % 0.041 0.033 −20.75 % < 0.01 < 0.01 −1.13 % 0.189 0.187 −1.03 % ER 2015 2019 Change 0.07 0.067 −4.83 % < 0.01 < 0.01 5.40 % 0.026 0.022 −16.79 % 0.01 0.01 −3.70 % 0.11 0.097 −7.66 % SR 2015 2019 Change 0.06 0.056 −7.94 % < 0.01 < 0.01 −9.26 % 0.026 0.027 1.52 % 0.01 0.01 −4.24 % 0.09 0.09 −5.01 % China 2015 2019 Change 0.60 0.612 1.91 % < 0.01 < 0.01 4.04 % 0.231 0.197 −14.53 % 0.05 0.048 −3.93 % 0.883 0.859 −2.74 % chemistry (UCX) mechanism (Eastham et al., 2014; Fisher et al., 2017) and were driven by the Goddard Earth Observing System Forward Processing (GEOS-FP) meteorological fields at a degraded horizontal resolution of 2◦ × 2.5◦ . The temporal resolutions are 1 h for surface meteorological variables and planetary boundary layer height (PBLH) and 3 h for other meteorological variables. The time steps used in the model are 10 min for transport and 20 min for chemistry and emissions, as recommend for the GEOS-Chem fullchemistry simulation at 2◦ × 2.5◦ (Philip et al., 2016). The nonlocal scheme for the boundary layer mixing process is described in Lin and McElroy (2010). Dry deposition was calculated by the resistance-in-series algorithm (Wesely, 1989; Zhang et al., 2001), and wet deposition followed that of Liu et al. (2001). The photolysis rates were available from the FAST-JX v7.0 photolysis scheme (Bian and Prather, 2002). All simulations were spun up for 1 year (July 2014 to July 2015) and output hourly mean C2 H6 VMR profiles globally ranging from the surface to 0.01 hPa at a horizontal resolution of 2◦ × 2.5◦ . This study only considered the C2 H6 simulations from 2015 to 2020 in the grid box containing Hefei (31.52–32.11◦ N by 116.53–118.02◦ E). In the recent past, the inventories led to significant underestimation of the C2 H6 simulation (e.g., HTAP2 in Franco et al., 2016). Since then, some efforts have improved the situation (e.g., Tzompa-Sosa et al., 2017). In this study, we refer to Sun et al. (2020a) for more details on the implemen- Atmos. Chem. Phys., 21, 11759–11779, 2021 tation of emission inventories. Briefly, global anthropogenic emissions were from the Community Emissions Data System (CEDS) inventory which is overwritten in Asia by the latest Multi-resolution Emission Inventory for China (MEIC) (Hoesly et al., 2018; Li et al., 2017; Lu et al., 2019; Zheng et al., 2018). Global biomass burning and biogenic emissions were from the Global Fire Emissions Database (GFED v4) inventory (Giglio et al., 2013) and the Model of Emissions of Gases and Aerosols from Nature (MEGAN version 2.1) inventory (Guenther et al., 2012), respectively. The CH4 emission fields are prescribed based on NOAA measurements for 1983–2016 and are extended to 2020 using the linear extrapolation of local 2011–2016 change rates (Murray, 2016; Lu et al., 2019). Particular improvements have been made for the latest bottom-up MEIC emission inventory with respect to the accuracy of vehicle emission modeling (Zheng et al., 2014), power plant emission calculation (Liu et al., 2015), and the non-methane VOC (NMVOC) speciation method (Li et al., 2014). Many studies have verified that the MEIC emission inventory can reasonably represent the anthropogenic emissions over Asia (Hoesly et al., 2018; Li et al., 2017; Lu et al., 2019; Y. Sun et al., 2021; Tian et al., 2018; Yin et al., 2020, 2019; Zheng et al., 2018). Anthropogenic C2 H6 emissions in China by region and category for the 2015 and 2019 MEIC emission inventories are summarized in Table 2. All subdivided geographical regions are shown in Fig. 2, and the re- https://doi.org/10.5194/acp-21-11759-2021 Y. Sun et al.: Reduction in C2 H6 from 2015 to 2020 over Hefei 11765 Table 3. GEOS-Chem model configurations and delimitations of all geographical regions used in sensitivity simulations. FF refers to fossil fuel, BVOCs refer to biogenic volatile organic compounds, BB denotes biomass burning, and BIOF refers to biofuel. Simulation Region Description BASE Global Standard simulation with all anthropogenic and natural C2 H6 emissions. The BASE simulation is taken as the reference and used for model evaluation noFF Global Turn off global fossil fuel C2 H6 emissions in the BASE simulation noBVOC Global Turn off global biogenic C2 H6 emissions in the BASE simulation noBB Global Turn off global biomass burning C2 H6 emissions in the BASE simulation noBIOF Global Turn off global biofuel C2 H6 emissions in the BASE simulation Rest noWR Global 27.6◦ N–48.8◦ N, 78.6◦ E–103.4◦ E Difference between BASE and the sum of FF, BVOC, BB, and BIOF contributions Turn off fossil fuel, biogenic, biomass burning, and biofuel C2 H6 emissions within western China (WR), i.e., region À in Fig. 2, in the BASE simulation noNR 34.6◦ N–53.5◦ N, 103.4◦ E–129.8◦ E Turn off fossil fuel, biogenic, biomass burning, and biofuel C2 H6 emissions within northern China (NR), i.e., region Á in Fig. 2, in the BASE simulation noCR 27.6◦ N–34.6◦ N, 103.4◦ E–115.6◦ E Turn off fossil fuel, biogenic, biomass burning, and biofuel C2 H6 emissions within central China (CR), i.e., region  in Fig. 2, in the BASE simulation noER 21.0◦ N–34.6◦ N, 115.6◦ E–123.6◦ E Turn off fossil fuel, biogenic, biomass burning, and biofuel C2 H6 emissions within eastern China (ER), i.e., region à in Fig. 2, in the BASE simulation noSR 21.0◦ N–27.6◦ N, 98.1◦ E–115.6◦ E Turn off fossil fuel, biogenic, biomass burning, and biofuel C2 H6 emissions within southern China (SR), i.e., region Ä in Fig. 2, in the BASE simulation ROW Rest of world Difference between BASE and the sum of WR, NR, CR, ER, and SR contributions sulting delimitations are summarized in Table 3. The delimitations of these geographical regions are based on the levels of urbanization and industrialization in China. Region À in Fig. 2 only covers a few sparse city clusters, representing the region with lowest population and least industrialization in China (Lu et al., 2019). Regions Á, Ã, and Ä cover the North China Plain (NCP), the Yangtze River Delta (YRD), and the Pearl River Delta (PRD) city clusters, respectively, which are the three most developed city clusters facing severe air pollution in China. Region  covers the Sichuan Basin (SCB) and central Yangtze River (CYR) city clusters, representing newly emerging severe air pollution in China. Total annual Chinese anthropogenic emissions of C2 H6 in 2015 and 2019 are 0.883 and 0.859 Tg, respectively. In both years, anthropogenic C2 H6 emissions in China are dominated by industrial and residential emissions. The highest anthropogenic C2 H6 emission rates (calculated as the ratio of total C2 H6 emission to the coverage) are in the densely populated and industrialized clusters in the eastern part of China (including northern China (NR), eastern China (ER), central China (CR), southern China (SR), and the adjacent regions) with large seasonal variation (Fig. S1 in the Supplement). The highest anthropogenic C2 H6 emissions typically occur in winter (Fig. S1). The anthropogenic emissions in the WR region are typically lower than those in other parts of China https://doi.org/10.5194/acp-21-11759-2021 because of a lower population and less industry in the region (Lu et al., 2019; Zheng et al., 2018). In order to quantify the contributions of different source categories and regions to the observed C2 H6 , we first conducted a reference full-chemistry simulation (BASE) with the implementation of all emission inventories as described above. We then conducted a series of sensitivity simulations to assess the change in each sensitivity simulation relative to the BASE simulation. Model configurations in this study were similar to those in Y. Sun et al. (2021) but with a different emission perturbation method. When an emission inventory was shut off in Y. Sun et al. (2021), the emissions of all atmospheric compounds in that inventory were suppressed. In contrast, this work only suppressed C2 H6 in each case except for biogenic and biomass burning emission perturbations, which suppressed all atmospheric compounds as we cannot separate C2 H6 emission from current biogenic and biomass burning emission inventories. Model configurations in this study are summarized in Table 3 and were described as follows: i. To analyze the contributions of different emission categories, we shut off C2 H6 in each individual emission inventory to evaluate the change in the simulation in the presence of C2 H6 in other emission inventories. As a result, the relative contribution of each emission category was estimated as the relative difference between Atmos. Chem. Phys., 21, 11759–11779, 2021 11766 Y. Sun et al.: Reduction in C2 H6 from 2015 to 2020 over Hefei the GEOS-Chem simulation in the presence and absence of C2 H6 in that emission inventory. We have conducted four such sensitivity simulations by shutting off C2 H6 emissions in (1) the fossil fuel emission inventory (including emissions from agriculture, industry, power plant, residential, and transport), (2) the biogenic emission inventory, (3) the biomass burning emission inventory, and (4) the biofuel emission inventory (Table 3). The sum of fossil fuel and biofuel C2 H6 emissions is defined as an anthropogenic C2 H6 source, and the sum of biogenic and biomass burning C2 H6 emissions are referred to as a natural C2 H6 source. ii. To analyze the contributions of different geographical regions, we shut off all categories of C2 H6 emissions (i.e., the aforementioned anthropogenic and natural C2 H6 sources) within each geographical region to assess the change in the simulation in the presence of C2 H6 emissions outside that geographical region. Thus, the relative contribution of each geographical region was estimated as the relative difference between the GEOS-Chem simulation in the presence and absence of C2 H6 emissions within that geographical region. We have conducted five such sensitivity simulations by shutting off C2 H6 emissions within five geographical regions, as shown in Fig. 2. 2.3 Generalized additive models (GAMs) regression In this study, we investigate the dependencies of C2 H6 on meteorological and emission factors using GAMs regression (Wood and Simon, 2004; Wood, 2004). Regression analysis is proceeded using the thin plate smoothing spline function (Pearce et al., 2011). Smoothing parameters and confidence intervals are calculated according to the restricted maximum likelihood standard (REML) and the unconditional Bayesian method, respectively (Pearce et al., 2011). GAMs regression is better than the traditional statistical models in dealing with nonlinear fittings (Veaux and Richard, 2012). For climate change applications, where there are many nonlinear relationships between variables, GAMs regression is particularly attractive (Zhang et al., 2019). We introduced a variety of potential meteorological and emission factors into the GAMs regression one at a time and performed significance tests based on the Akaike information criteria (AIC) values (Wood and Simon, 2004). The explanatory variables which passed the significance tests with the smallest AIC values were included into the final GAMs model. Furthermore, explanatory variables in GAMs regression may interact with each other and result in unstable fittings due to the internal multicollinearity. For the explanatory variables that show a strong collinearity with each other, we only included one of them in the final GAMs model. The degree of multicollinearity can be quantified by the variance inflation factor (VIF) (Ma et al., 2020). Generally, a stronger Atmos. Chem. Phys., 21, 11759–11779, 2021 collinearity between the explanatory variables results in a larger VIF, and the VIF of an explanatory variable could be 1.0 if it is not correlated with other explanatory variables (Ma et al., 2020). In this study, we included all of the meteorological and emission factors in the GAMs and calculated the VIF for all of the influencing factors. The multicollinearity diagnosis concluded that the main causes of multicollinearity are between the HCN and CO and between the tropopause height and the PBLH. Including either of these two data pairs in the GAMs regression showed significant collinearity with VIF values of greater than an empirical threshold of 4.0, indicating an unstable regression (Lin et al., 2018). After omitting HCN and PBLH in the final GAMs model, the adjusted VIF values of all the variables were less than 4.0 and the variables uniformly passed the significance tests. As a result, the final GAMs model in the context of the C2 H6 troDMF time series y can be described as follows (Pearce et al., 2011): log(y) = β + S(ua) + S(va) + S(omega) + S(qv) + S(troph) + S(pres) + S(temp) + S(CH4 ) + S(CO) + ε, (1) where β and ε are the mean response constant and the fitting residual, respectively. S(ua), S(va), S(omega), S(qv), S(troph), S(pres), S(temp), S(CH4 ), and S(CO) are the smoothing functions of daily average zonal wind (m s−1 ), meridional wind (m s−1 ), vertical wind (Pa s−1 ), water vapor concentration (%), tropopause height (km), pressure (hPa), temperature (◦ C), CH4 troDMF (ppbv), and CO troDMF (ppbv). Positive values of ua, va, and omega represent northward, eastward, and upward winds, respectively. The sum of S(CH4 ) and S(CO) is referred to as the emission influences, and the sum of the remaining smoothing functions is referred to as the meteorological influences. To drive the GAMs regression, we first derived CH4 and CO VMR profiles from direct solar absorption spectra similar to that of C2 H6 (see Sect. 2.1). The spectra for CH4 retrievals are exactly the same as those of C2 H6 , but the spectra for CO are saved at a different filter channel. The respective VMR profiles were then converted to troDMF values following the method of C2 H6 . The retrieval configurations, wave band selections and the interfering gases considerations for CH4 and CO can be found in Sun et al. (2018b). The DOFS of the retrievals between the surface and 10 km altitude for both CH4 and CO are larger than 1.5, and the corresponding retrieval errors are less than 8 % (Sun et al., 2018b). All meteorological factors are from the GEOS-FP meteorological fields at their native resolution of 0.25◦ × 0.3125◦ ranging from the surface to 0.01 hPa at a temporal resolution of 1 h. As the meteorological fields and C2 H6 concentration are not uniformly distributed along the altitude, the summing mean values of the meteorological fields cannot properly characterize the meteorological influences. In this study, we use a method similar to that of Shaiganfar et al. (2017) to increase the influence weighting toward the lower troposphere. As a result, all https://doi.org/10.5194/acp-21-11759-2021 Y. Sun et al.: Reduction in C2 H6 from 2015 to 2020 over Hefei meteorological parameters except tropopause height (troph) are converted into the C2 H6 profile weighting averaged value ωavg through Eq. (2): P ω(zi ) · c(zi ) · Air mass(zi ) , (2) ωavg = i P i c(zi ) · Air mass(zi ) where ω(zi ), c(zi ), and Air mass(zi ) represent the value of the meteorological factor, C2 H6 concentration, and the air mass at the altitude zi , respectively. 3 Variability and comparison with GEOS-Chem model data We have compared the observed daily mean time series, seasonal cycle, and interannual variability of C2 H6 troDMF to the GEOS-Chem BASE simulations for investigating the chemical model performance for specific polluted regions over eastern China. As the vertical resolution of GEOSChem is different from the FTIR observation, smoothing correction has been carried out for the GEOS-Chem profiles (Rodgers and Connor, 2003). First, the GEOS-Chem daily mean profiles of C2 H6 have been interpolated to the FTIR altitude grid to ensure a common altitude grid. In order to match observations from the FTIR which only operates during daytime, the average for GEOS-Chem simulations was only performed during daytime from 09:00 to 17:00 LT (local time). The interpolated profiles were then smoothed by the concurrent seasonal mean values of the FTIR averaging kernels and a priori profiles (Rodgers, 2000; Rodgers and Connor, 2003). The smoothed GEOS-Chem profiles were subsequently converted to troDMF values using the corresponding regridded air density profiles from the model with the method described in Sect. 2.1. Finally, the GEOS-Chem C2 H6 troDMF time series only for the days with available FTIR observations were averaged by month and compared with the FTIR monthly mean data. Figure 3a shows the comparison of daily mean time series of C2 H6 troDMF between the FTIR observation and the smoothed GEOS-Chem model simulation from 2015 to 2020. Figure 3b compares the seasonal cycles derived from Fig. 3a for the days with available FTIR observations only. Generally, the measured features in terms of seasonality and interannual variability can be reproduced by the GEOS-Chem simulations with a correlation coefficient (r) of 0.88 (Fig. S2). Large GEOS-Chem vs. FTIR differences tended to occur in the trough and peak of the observations. For instance, the observed monthly mean value of C2 H6 troDMF was overestimated by 35.6 % (calculated as (GEOS-Chem − FTIR) / FTIR) in July and underestimated by 14.6 % in December by GEOS-Chem. These discrepancies may be mainly attributed to uncertainties in the horizontal transport and vertical mixing schemes simulated by the GEOS-Chem model at a relatively coarse spatial resolution, which are difficult to match column observation over https://doi.org/10.5194/acp-21-11759-2021 11767 a single point. In addition, the number of C2 H6 measurements at Hefei on each measurement day varied a wide range from 1 to 17 depending on the weather conditions, but GEOS-Chem simulations are available consecutively by hour for the same day. This difference in the temporal resolution of GEOS-Chem and FTIR could also cause inconsistencies due to the high variability of atmospheric C2 H6 . However, considering the concurrent data pairs only (± 30 min), the averaged difference between GEOS-Chem and FTIR data (GEOS-Chem − FTIR) is (−0.02 ± 0.05) ppbv (−1.6 ± 4.2) %, which is within the FTIR uncertainty budget. As a result, GEOS-Chem can simulate the C2 H6 concentration and variability for specific polluted regions over eastern China. With improved emission inventories, previous studies have also found that global chemistry transport models were able to reproduce the absolute values as well as seasonal cycles of the ground-based FTIR C2 H6 observations in other parts of the world (Franco et al., 2015, 2016; Tzompa-Sosa et al., 2017). As typically observed, the peak-to-peak amplitude of the modulation with respect to the monthly mean of C2 H6 troDMF spanned a large range from −16.0 % to 72.8 % depending on season (Fig. 3b). The observed C2 H6 troDMF roughly decreases over time for the first half of the year and increases over time for the second half of the year (Fig. 3b). High levels of C2 H6 troDMF occur in the late autumn to early spring, and low levels of C2 H6 troDMF occur in late spring to early autumn. The observed C2 H6 troDMF reached a minimum monthly mean value of (0.36 ± 0.26) ppbv in July and a maximum monthly mean value of (1.76 ± 0.35) ppbv in December. C2 H6 troDMFs in December were on average 4.9 times higher than those in July. As the tropospheric OH oxidation capability in summer is higher than that in winter, the C2 H6 seasonality characterized by a winter maximum and a summer minimum was also observed in other FTIR stations (Angelbratt et al., 2011; Franco et al., 2015, 2016; Lutsch et al., 2019; Nagahama and Suzuki, 2007; Rinsland et al., 2002; Simpson et al., 2012; Viatte et al., 2015, 2014; Vigouroux et al., 2012; Zeng et al., 2012; Zhao et al., 2002). We have used the bootstrap resampling method of Gardiner et al. (2008) to evaluate the seasonality and interannual variability of C2 H6 troDMF, where a third Fourier series and a linear function were used to fit daily mean time series of C2 H6 troDMF from both FTIR observations and GEOSChem model simulations. We incorporated the errors arising from the autocorrelation in the residuals into the uncertainties in the change rates following the procedure of Santer et al. (2008). The observed C2 H6 troDMFs from 2015 to 2020 showed a negative change rate of (−2.60 ± 1.34) % yr−1 , which is in reasonable agreement with the modeled change rate of (−2.1 ± 0.7) % yr−1 (Fig. 3a). Atmos. Chem. Phys., 21, 11759–11779, 2021 11768 Y. Sun et al.: Reduction in C2 H6 from 2015 to 2020 over Hefei Figure 3. (a) C2 H6 troDMF time series comparison between the FTIR observation and GEOS-Chem model BASE simulation from 2015 to 2020 over Hefei, eastern China. The seasonality and interannual variability are represented by red dots and a black line, respectively, which are fitted using a bootstrap resampling model with a third Fourier series and a linear function. (b) Seasonal variations in C2 H6 troDMF by FTIR and GEOS-Chem simulation. The bold curves and the shadows are the monthly mean values and the 1σ standard variations, respectively. Vertical error bars for FTIR and GEOS-Chem represent retrieval uncertainties and diurnal variabilities, respectively. A retrieval error of 6.21 % in Table 1 was used to estimate the absolute uncertainties of all observations. As a result, the absolute uncertainties in winter are larger than those in summer due to a higher C2 H6 level in the season. 4 GAMs regression results and interpretation C2 H6 troDMF time series from 2015 to 2020 over Hefei from the FTIR and the GAMs regression model are shown in Fig. 4. The observed C2 H6 variability can be reproduced by the GAMs regression model with good agreement, as confirmed by a correlation coefficient (r) of 0.90 (Figs. 4a, S3). Meanwhile, the observed C2 H6 troDMF time series also show high correlations with both the accumulated meteorological factor (r = 0.88) (Fig. 4b) and the accumulated emission factor (r = 0.70) (Fig. 4c), indicating that both meteorological and emission influences are important factors for driving the C2 H6 troDMF variability. Generally, both meteorological and emission factors show positive influences on C2 H6 troDMFs in the cold season (December–January– February/March–April–May, DJF/MAM) and negative influences on C2 H6 troDMFs in the warm season (June–July– August/September–October–November, JJA/SON). However, the seasonal dependency of meteorological influence is stronger than that of emission influence. During the studied years, the year-to-year differences in meteorological influence are small, and the positive emission influence showed an overall decreasing change rate from 2016 onward, which has Atmos. Chem. Phys., 21, 11759–11779, 2021 probably driven a decreasing change rate in C2 H6 troDMF in recent years. The influence of each explanatory variable xi in GAMs regression, calculated as 100 % · [es (xi ) − 1], is shown in Fig. 5, which reflects the influence of each individual variable on the relative change in C2 H6 troDMF. The corresponding DOFS of each smoothing function are also shown in Fig. 5. If an explanatory variable is linearly correlated with C2 H6 troDMF, the DOFS of the resulting smoothed variable could be equal to 1, and the larger the slope, the higher the linear response. Otherwise, the larger the deviation of DOFS relative to 1, the more significant the nonlinear relationship (Veaux and Richard, 2012). During the studied years, the DOFS of zonal wind (ua), convection wind (omega), pressure (pres), tropopause height (troph), temperature (temp), and CO troDMF (co) were close to 1, reflecting a roughly linear relationship of these explanatory variables with C2 H6 troDMF. In contrast, the DOFS of meridional wind (va), H2 O troDMF (qv), and CH4 troDMF (CH4 ) were much greater than 1, reflecting a significant nonlinear relationship with C2 H6 troDMF. The observed C2 H6 troDMF was influenced by many factors. The zonal wind (ua), meridional wind (va), CH4 troDMF (CH4 ), and CO troDMF (co) showed positive inhttps://doi.org/10.5194/acp-21-11759-2021 Y. Sun et al.: Reduction in C2 H6 from 2015 to 2020 over Hefei 11769 Figure 4. (a) C2 H6 troDMF time series from 2015 to 2020 over Hefei, eastern China, from FTIR and the GAMs regression model. (b) Time series of accumulated meteorological smooth functions, S(meteos); (c) time series of accumulated emission smooth functions, S(non-meteos). Positive and negative influences are indicated using red and blue bars, respectively. Correlation coefficients for the total, meteorological, and emission influences are also shown. Figure 5. The influence of each individual variable in the GAMs model on C2 H6 troDMF from 2015 to 2020 over Hefei, eastern China. Panels (a)–(i) are for zonal wind (ua), meridional wind (va), convection wind (omega), pressure (pres), tropopause height (troph), H2 O troDMF (qv), temperature (temp), CH4 troDMF (CH4 ), and CO troDMF (co), respectively. The DOFS of each smoothing function is included in the parenthetical information in each panel heading. The x axis represents the variation range of each variable, and the y axis represents the relative percentage change in C2 H6 troDMF relative to its annual mean value. https://doi.org/10.5194/acp-21-11759-2021 Atmos. Chem. Phys., 21, 11759–11779, 2021 11770 Y. Sun et al.: Reduction in C2 H6 from 2015 to 2020 over Hefei Figure 6. Absolute (a) and relative (b) contributions of fossil fuel, biogenic, biomass burning, and biofuel emission sources to the observed C2 H6 variability from 2015 to 2020 over Hefei, eastern China. The remaining contribution, calculated as the difference between the BASE simulation and the sum of anthropogenic and natural contributions, is also shown. All contributions are grouped by season. Vertical error bars represent 1σ standard variation. fluences on the observed C2 H6 troDMF variability, whereas the other explanatory variables showed negative influences. The results show that the northward, eastward, and downward convection winds facilitate the accumulation of C2 H6 over the observation site and result in higher C2 H6 troDMF. As most anthropogenic and biomass burning sources of CO as well as fossil fuel sources of CH4 are common sources of C2 H6 , C2 H6 troDMF gradually went up as CH4 and CO troDMFs increased. In contrast, meteorological conditions such as high temperature, high humidity, and low pressure are more favorable to C2 H6 oxidation, resulting in lower C2 H6 troDMF. Meanwhile, deep upward convective winds and high tropopause height facilitate the removal of C2 H6 over the observation site and result in lower C2 H6 troDMF. 5 5.1 Source attribution Contributions of different source categories and regions The absolute and relative seasonal contributions of fossil fuel, biogenic, biomass burning, and biofuel emissions to C2 H6 variability from 2015 to 2020 over Hefei are shown in Fig. 6. The GEOS-Chem annual mean C2 H6 troDMF simulations were decreased by 0.51, 0.27, 0.32, and 0.20 ppbv in the absence of fossil fuel, biogenic, biomass burning, and biofuel C2 H6 emission inventories, which correspond to Atmos. Chem. Phys., 21, 11759–11779, 2021 34.6 %, 18.4 %, 21.3 %, and 13.5 % of the relative contribution to the modeled C2 H6 variability, respectively. The anthropogenic emissions account for 48.1 % of the C2 H6 variability, and the natural emissions account for 39.7 %. The remaining, contribution calculated as the difference between the BASE simulation and the sum of anthropogenic and natural contributions, is 0.17 ppbv (12.2 %). This missing contribution can be largely attributed to nonlinear interactional effects among different sources which were not captured by the sensitivity simulations. Indeed, shutting off an emission inventory may induce significantly lower concentrations in atmospheric compounds (i.e., C2 H6 for noFF and noBIOF or all suppressed compounds for noBVOC and noBB simulations; see Table 3 for an explanation of these terms) globally. On the one hand, some of them may react with OH, which would lead to higher OH concentrations being available for the oxidation of C2 H6 , eventually enhancing the C2 H6 destruction from other emission categories. On the other hand, some of them may form OH by a series of oxidation reaction, which would lead to lower OH concentrations being available for the oxidation of C2 H6 , eventually mitigating the C2 H6 destruction from other emission categories. However, it is difficult to quantify the nonlinear impact of each individual emission category, as the concentration and spatial distribution of C2 H6 in each emission category are different. Especially when biogenic and biomass burning emissions are suppressed, the impacts become hard to assess, as all NMVOC compounds play a key role in both OH formahttps://doi.org/10.5194/acp-21-11759-2021 Y. Sun et al.: Reduction in C2 H6 from 2015 to 2020 over Hefei 11771 Figure 7. Absolute (a) and relative (b) contributions of ER, CR, NR, WR, SR, and ROW regions to the observed C2 H6 variability from 2015 to 2020 over Hefei, eastern China. All contributions are grouped by season. The geographical definition of each region is summarized in Table 3. Vertical error bars represent 1σ standard variation. tion and destruction. Investigating the nonlinear impact of each individual emission category would require additional work that is beyond the scope of the present study. The contributions of all emission sources are dependent on season, and the fossil fuel contribution shows the largest seasonal difference, which consolidates the GAMs regression results that the emission influences are seasonally dependent. The fossil fuel contributions in winter and spring (DJF/MAM) are larger than those in summer and autumn (JJA/SON), with a maximum of 52.0 % in DJF and a minimum of 13.0 % in JJA. The JJA/SON meteorological conditions which show stronger solar radiation, higher temperature, wetter atmospheric condition, and lower pressure than those in DJF/MAM are more favorable for increasing VOC emissions from biogenic sources (BVOCs), which consolidates the fact that C2 H6 abundance from biogenic source in JJA/SON is larger than that in DJF/MAM. The missing remainder contributes to a maximum of 32 % in JJA when the C2 H6 oxidation reaches the seasonal maximum and is, thus, more sensitive to the on–off state of different sources. Figure 7 explores the absolute and relative seasonal contributions of the ER, CR, NR, WR, and SR regions to the C2 H6 variability from 2015 to 2020 over Hefei. The GEOS-Chem annual mean C2 H6 troDMF simulations were decreased by 0.28, 0.22, 0.29, 0.07, and 0.12 in the absence of C2 H6 emissions in the ER, CR, NR, WR, and SR regions, which correspond to 21.5 %, 15.8 %, 20.3 %, 5.7 %, and 8.9 %, of the relative contribution to the modeled C2 H6 variability, respec- https://doi.org/10.5194/acp-21-11759-2021 tively. The contributions of all geographical regions are also seasonally dependent. The results show that the observed C2 H6 variability was largely attributed to emissions within China (74.1 %), which show a maximum in JJA/SON and a minimum in DJF/MAM. Due to their vicinity to the observation site, the ER, CR, and NR regions dominated the contribution within China (57.6 %). The remaining contribution, calculated as the difference between the BASE simulation and the total contributions of above individual source regions, is 0.42 ppbv (25.9 %). This contribution is the sum of the C2 H6 emissions outside China (ROW – rest of the world) and the nonlinear interactional effects among the geographical sensitivity simulations. This remaining contribution in DJF/MAM is ∼ 4.0 times larger than that in JJA/SON. Considering that the nonlinear interactional effects mainly occur in JJA/SON, but this remaining contribution shows a seasonal minimum value in the meantime, the remaining contribution can be largely attributed to ROW contributions. As a relatively long-lifetime species (a few months), C2 H6 emissions originating from either nearby or in distant areas can be transported to the Hefei site under favorable weather conditions and, thus, contribute to the observed C2 H6 variability. In addition, atmospheric compounds originating either nearby or in distant areas, which affect the chemistry of C2 H6 oxidation, could also affect the observed C2 H6 variability. For contributions within China, the lowest contribution of the WR region to the observed C2 H6 variability is largely attributed to the lowest C2 H6 emission rates in this Atmos. Chem. Phys., 21, 11759–11779, 2021 11772 Y. Sun et al.: Reduction in C2 H6 from 2015 to 2020 over Hefei Figure 8. Spatial distribution of C2 H6 troDMF in the GEOS-Chem BASE simulations in different seasons. The arrows indicate horizontal wind vectors at 900 hPa; the observation site is marked with a yellow dot. Meteorological fields are from the GEOS-FP 0.25◦ × 0.3125◦ dataset. region (Table 2). The smaller contribution of the SR region to the observed C2 H6 variability in DJF/MAM in comparison with the ER, CR, and NR regions is mainly attributed to fewer air masses that originated in south China under the dry winter monsoon conditions (see Sect. 5.2). 5.2 Transport inflow and outflow pathways The direct GEOS-Chem sensitivity simulations can clearly characterize the 3D transport inflow and outflow pathways of C2 H6 over the observation site. Figure 8 shows the spatial distribution of the GEOS-Chem C2 H6 BASE simulations around China along with horizontal wind vectors at 900 hPa in different seasons. General atmospheric circulation patterns over eastern China are typically affected by midlatitude westerlies and the Asian monsoon, including the East Asian summer monsoon and the South Asian summer monsoon (Chen et al., 2009; Liang et al., 2005; Liu et al., 2003). Figure 9 illustrates the latitude–height distributions of the C2 H6 VMR over China from six source regions along with the 3D atmospheric circulation patterns in different seasons. Thus, the 3D transport inflow and outflow pathways of C2 H6 over the observation site are deduced as follows. As indicated by the arrows in Figs. 8 and 9, the highpressure system over the Eurasian continent in DJF triggers the descent of very cold air over eastern China and results in Atmos. Chem. Phys., 21, 11759–11779, 2021 air masses converging toward the observation site from the western and northern areas, while the high-pressure system over the Indian Ocean and the Pacific in JJA triggers the ascent of strongly heated air over eastern China and results in air masses converging toward the observation site from South Asia and East Asia (SEAS) (Liang et al., 2004; Liu et al., 2003). In DJF, the midlatitude westerlies extend to the tropics (about 15◦ N) over the middle and upper troposphere and to the subtropics (about 30◦ N) near the surface, while the easterlies mainly prevail in the tropics and are weak over eastern China (Figs. 8, 9). In the summer monsoon season, the atmospheric circulation patterns over eastern China change dramatically and are dominated by a surface wind regime originating from the Pacific, the South China Sea, or the Arabian Sea (Figs. 8, 9). Meanwhile, the midlatitude westerlies recede to the north temperate zone (north of 30◦ N) and the westerly jet center shifts to north of 50◦ N in JJA (from ∼ 30◦ N in DJF) (Fig. 8). In JJA, the tropical region is characterized by strong easterlies in the upper troposphere and by southwesterly air flow in the lower troposphere (Fig. 9). The prevailing winds in the transition seasons in MAM and SON are still westerlies with frequent cold fronts (Figs. 8, 9). These abovementioned seasonal circulation patterns determine the transport inflow and outflow of C2 H6 around the observation site. However, the transport scales are also influ- https://doi.org/10.5194/acp-21-11759-2021 Y. Sun et al.: Reduction in C2 H6 from 2015 to 2020 over Hefei 11773 Figure 9. The first row shows the latitude–height distributions of the C2 H6 VMR averaged over 113–121◦ E in spring (MAM), originating from different source regions (corresponding to different columns). See Table 3 for latitude and longitude definitions. The white area indicates topography, and the white contours at intervals of 6 m s−1 are the easterly (dashed) and westerly (solid) mean meridional winds; the wind vectors (consisting of zonal wind in m s−1 and vertical velocity in units of Pa s−1 ) are represented by arrows; the observation site is marked with a yellow line. The second to fourth rows are the same as the first row but for summer (JJA), autumn (SON), and winter (DJF), respectively. Meteorological fields are from the GEOS-FP 0.25◦ × 0.3125◦ dataset. enced by source location and strength, travel trajectory, and travel time (Liu et al., 2003). Generally, C2 H6 emissions from the CR, NR, and WR regions can be transported to the observation site by strong westerlies throughout the year (Fig. 9). C2 H6 from the SR region can be transported to the observation site by deep convection followed by northward transport into the midlatitude westerlies in MAM or driven by the South Asian summer monsoon or westerlies in other seasons (Liu et al., 2003) (Fig. 8). The observed C2 H6 transport inflow originating from the local ER region is mainly driven by the local circulation cell or Asian monsoon. The driver for C2 H6 transport inflow originating from the northern ROW (> 32◦ N) is the same as those from the CR, NR, and WR regions. The driver for C2 H6 transport inflow originating from the southern ROW (< 32◦ N) is the same as that from the SR region. However, C2 H6 originating from ROW relative to those from China reaches a higher altitude due to longer transport distances. The fact that the ROW contributions in JJA/SON are lower than those in DJF/MAM can be partly attributed to stronger removal along the long-range transport pathway by https://doi.org/10.5194/acp-21-11759-2021 abundant wet precipitation and oxidation during the summer monsoon and post-monsoon season. Seasonal variability in C2 H6 transport outflow over the observation site is mainly associated with the monsoon system. In DJF, C2 H6 over the observation site is capped in the lower troposphere by the subsidence over eastern China and is swept southwestward by northeasterly winds into southwestern areas, where it is lifted up into the free troposphere by convection and then flows away northeastward (Fig. 9). In JJA, the observed C2 H6 is transported northeastward by the Asian monsoon and undergoes frequent lifting into the upper troposphere by deep convection (Figs. 8, 9). Frequent cold fronts are common phenomena during meteorological transitional periods in MAM and SON. In SON, the winter monsoon builds a continental high system, and sweeps the observed C2 H6 in the lower troposphere northward to relatively high latitudes where it can be lifted up into the free troposphere by deep convection or cold fronts. In MAM, convection at lower latitudes over the Asian continent starts to rise, which lifts the observed C2 H6 up into the free troposphere before it then flows away southward. Atmos. Chem. Phys., 21, 11759–11779, 2021 11774 5.3 Y. Sun et al.: Reduction in C2 H6 from 2015 to 2020 over Hefei Potential factors driving interannual variability of C2 H 6 China has implemented a series of active clean air policies since 2013 to mitigate severe air pollution problems (Sun et al., 2020; Zhang et al., 2019; Zheng et al., 2018). Since then, the emissions of major air pollutants have decreased, and the overall air quality has greatly improved (Sun et al., 2020; Zhang et al., 2019; Zheng et al., 2018). Many air pollutants, such as NO2 , sulfur dioxide (SO2 ), particulate matter with an aerodynamic diameter of less than 2.5 µm (PM2.5 ), PM10 , CO, black carbon (BC), and organic carbon (OC), showed negative trends in recent years (Lu et al., 2019; Zhang et al., 2019; Zheng et al., 2018). We follow the method of Zheng et al. (2018) to estimate the relative change rate of anthropogenic C2 H6 emissions in China during the 2015– 2019 period using the MEIC emission inventory. As tabulated in Table 2, anthropogenic C2 H6 emissions in all geographical regions showed a decreasing change rate during 2015–2019 except those in the WR region where industrialization, urbanization, land use, and infrastructure construction have expanded rapidly in recent years, resulting in an increasing change rate of anthropogenic C2 H6 emissions in the region (Ran et al., 2014). The relative change rates of anthropogenic C2 H6 emissions in China during the 2015– 2019 period are estimated as 12.12 % for WR, −5.32 % for NR, −1.03 % for CR, −7.66 % for ER, −5.01 % for SR, and −2.74 % in total. The major driving forces for the decline in C2 H6 emissions over China are attributed to the reductions from residential and transport sectors, with relative change rates of −14.53 % and −3.93 %, respectively (Table 2). The interannual change rate of anthropogenic C2 H6 emissions in China in recent years is estimated to be −0.69 % yr−1 , calculated as −2.74 % / (2019–2015), which is much lower than the observed decreasing change rate in C2 H6 troDMF (−2.60 ± 1.34 % yr−1 ), indicating that additional driving forces could exist (e.g., reductions in natural C2 H6 emissions in China or reductions in long-range transport of C2 H6 emissions originating from either anthropogenic or natural sources outside of China). On the one hand, the Law of the People’s Republic of China on the Prevention and Control of Atmospheric Pollution included the prohibition of crop residue burning over China in 2015 because crop residue burning emissions can result in poor air quality (http://www.chinalaw.gov.cn, last access on 19 June 2020). Since then, crop residue burning events over China have decreased dramatically (Sun et al., 2020). Meanwhile, biomass burning events in Africa, SEAS, and the Oceania regions in 2015 were higher than those in the following years due to the El Niño–Southern Oscillation (ENSO) (Sun et al., 2020). The decrease in global biomass burning emissions could also have contributed to the observed decreasing change rate in C2 H6 troDMF over Hefei since 2015. On the other hand, large oil price fluctuations in recent years have probably tightened oil and gas development around the globe Atmos. Chem. Phys., 21, 11759–11779, 2021 which can cause a reduction in C2 H6 leakage worldwide. However, C2 H6 observations around the globe and more statistical data are needed to support this deduction, which is beyond the scope of this paper and requires further study. 6 Conclusions Ethane (C2 H6 ) is an important greenhouse gas and plays a significant role in tropospheric chemistry and climate change. As a relatively long-residence-time species (a few months), observations of C2 H6 can reflect regional and hemispheric changes in emissions and climate and can be assimilated into a chemical transport model to assess nonlocal emissions and provide valuable insights into model biases of C2 H6 simulations. This study, for the first time, presents and quantifies the variability, sources, and transport of C2 H6 over densely populated and highly industrialized eastern China using groundbased high-resolution Fourier transform infrared (FTIR) observations. Seasonal and interannual variabilities of C2 H6 over Hefei, eastern China, from 2015 to 2020 have been investigated. The dependencies of C2 H6 on meteorological and emission factors were analyzed using generalized additive models (GAMs). For the first time, FTIR C2 H6 time series are applied to evaluate the standard GEOS-Chem fullchemistry model with respect to the simulation of C2 H6 for specific polluted regions over eastern China. GEOS-Chem model simulation with the state-of-the art inventory is in good agreement with the FTIR observation. The GEOSChem model was further run in a sensitivity mode to quantify the relative contribution of various source categories and regions to the observed C2 H6 variability. The threedimensional (3D) transport inflow and outflow pathways of C2 H6 over the observation site were finally determined by the GEOS-Chem sensitivity simulation and the analysis of the meteorological fields. We obtained a retrieval error of 6.21 ± 1.2 (1σ )% and degrees of freedom (DOFS) of 1.47 ± 0.2 (1σ ) in retrieval of the C2 H6 tropospheric column-averaged dry-air mole fraction (troDMF). The observed C2 H6 troDMF reached a minimum monthly mean value of (0.36 ± 0.26) ppbv in July and a maximum monthly mean value of (1.76 ± 0.35) ppbv in December, and showed a negative change rate of (−2.60 ± 1.34) % yr−1 from 2015 to 2020. Generally, both meteorological and emission factors have positive influences on C2 H6 troDMF in the cold season (DJF/MAM) and negative influences in the warm season (JJA/SON). GEOSChem model sensitivity simulations concluded that the anthropogenic (fossil fuel and biofuel) emissions accounted for 48.1 % of C2 H6 variability over Hefei, and the natural (biomass burning and biogenic) emissions accounted for 39.7 %. The observed C2 H6 variability over Hefei was mainly attributed to the emissions within China (74.1 %), where central, eastern, and northern China dominated the https://doi.org/10.5194/acp-21-11759-2021 Y. Sun et al.: Reduction in C2 H6 from 2015 to 2020 over Hefei contribution (57.6 %). Seasonal variability in C2 H6 transport inflow and outflow over the observation site is largely related to the midlatitude westerlies and the Asian monsoon system. The reduction in C2 H6 from 2015 to 2020 mainly results from the decrease in local and transported C2 H6 emissions, which points to air quality improvement in China in recent years. This study can not only enhance insights into regional emission, transport, and air quality actions over China but can also contribute to form a new reliable remote sensing dataset in this sparsely monitored region for climate change research. Code and data availability. The new ground-based Fourier transform infrared (FTIR) spectroscopic remote sensing dataset for atmospheric C2 H6 over Hefei, eastern China, from this study can be accessed at https://doi.org/10.6084/m9.figshare.13020545 (Sun and Hao, 2020). The MEIC emission inventories used in this study are available from http://meicmodel.org/ (last access: 17 March 2021) (MEIC model Community, 2019). Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/acp-21-11759-2021-supplement. Author contributions. YS designed and wrote the paper with input from all coauthors. HY carried out the GEOS-Chem simulations and GAMs regression. BZ provided the latest MEIC emission inventory. CL, EM, JN, YT, XL, MP, WW, CS, QH, MQ, and YT contributed to this work by providing constructive comments. Competing interests. The authors declare that they have no conflict of interest. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements. The processing and post-processing environment for SFIT4 are provided by National Center for Atmospheric Research (NCAR), Boulder, Colorado, USA. The NDACC network is acknowledged for supplying the SFIT software. The LINEFIT code is provided by Frank Hase, Karlsruhe Institute of Technology (KIT), Institute for Meteorology and Climate Research (IMKASF), Germany. We thank the senate of Bremen, Germany, for support. We are grateful to the FTIR group at university of Wollongong, Australia, for help with setting up and operating the FTIR spectrometer at Hefei. We also thank the GEOS-Chem team and Tsinghua University, China, for providing the latest MEIC inventory. https://doi.org/10.5194/acp-21-11759-2021 11775 Financial support. This work is jointly supported by the National Key Research and Development Program of China (grant nos. 2019YFC0214802, 2017YFC0210002, 2016YFC0203302, 2018YFC0213201, 2019YFC0214702, and 2016YFC0200404), the National Science Foundation of China (grant nos. 41775025, 41575021, 51778596, 91544212, 41722501, and 51778596), and the Sino-German Mobility Programme (grant no. M-0036). Emmanuel Mahieu is a Senior Research Associate with the Fonds de la Recherche Scientifique – FNRS. His contribution has been primarily supported by the F.R.S.-FNRS (grant no. J.0147.18). Review statement. This paper was edited by Ilse Aben and reviewed by three anonymous referees. 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Research Evaluation, 30(3), 2021, 361–369 doi: 10.1093/reseval/rvab013 Advance Access Publication Date: 8 May 2021 Article Annette Boaz Alison O’Shea4 1, *, Robert Borst 2, Downloaded from https://academic.oup.com/rev/article/30/3/361/6272492 by guest on 18 May 2024 How far does an emphasis on stakeholder engagement and co-production in research present a threat to academic identity and autonomy? A prospective study across five European countries *, Maarten Kok3 and 1 Department of Health Services Research and Policy, Faculty of Public Health and Policy 15-17 Tavistock Place, London, WC1H 9SH, UK, 2Institute of Health Policy and Management, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands, 3Department of Health Sciences, Amsterdam Public Health Research Institute, Amsterdam, The Netherlands and 4Faculty of Health, Social Care and Education, Kingston University and St George’s, University of London, St George’s Campus, 6th Floor Hunter Wing, Cranmer Terrace, London SW17 0RE, UK *Corresponding author. Email: annette.boaz@lshtm.ac.uk (A.B.) Abstract There is a growing recognition that needs more to be done to ensure that research contributes to better health services and patient outcomes. Stakeholder engagement in research, including coproduction, has been identified as a promising mechanism for improving the value, relevance and utilization of research. This article presents findings from a prospective study which explored the impact of stakeholder engagement in a 3-year European tobacco control research project. That research project aimed to engage stakeholders in the development, testing and dissemination of a return-on-investment tool across five EU countries (the Netherlands, Spain, Hungary, Germany and the UK). The prospective study comprised interviews, observations and document review. The analysis focused on the extent to which the project team recognized, conceptualized and operationalized stakeholder engagement over the course of the research project. Stakeholder engagement in the European research project was conceptualized as a key feature of pre-designated spaces within their work programme. Over the course of the project, however, the tool development work and stakeholder engagement activities decoupled. While the modelling and tool development became more secluded, stakeholder engagement activities subtly transformed from co-production, to consultation, to something more recognizable as research participation. The contribution of this article is not to argue against the potential contribution of stakeholder engagement and co-production, but to show how even well-planned engagement activities can be diverted within the existing research funding and research production systems where nonresearch stakeholders remain at the margins and can even be seen as a threat to academic identify and autonomy. Key words: research utilization; stakeholder engagement; co-production; smoking cessation; return on investment; public health; health services research; academic identify C The Author(s) 2021. Published by Oxford University Press. V 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. 361 362 1. Research Evaluation, 2021, Vol. 30, No. 3 Introduction 2. Methods This article presents findings from a prospective study which explored the impact of stakeholder engagement in a 3-year tobacco control research project. Drawing on literature from science and technology studies that explore the role of stakeholders in research (Jasanoff 2004; Callon et al. 2009) and Henkel’s work on academic identity (Henkel 2005), the article focuses in particular on the extent to which the EQUIPT project team recognized, conceptualize and operationalize stakeholder engagement over the course of the study. The EQUIPT research project aimed to engage stakeholders in the development, testing and dissemination of a tobacco control returnon-investment tool across five EU countries (i.e. Netherlands, Spain, Hungary, Germany and the UK). Data collection of this study comprised interviews with the project team and stakeholders of the EQUIPT research project, observations of meetings and events involving these stakeholders and a review of documents relevant to the meetings and events. Our study design repeatedly adapted to the organization of the EQUIPT project to be able to study and reflect Downloaded from https://academic.oup.com/rev/article/30/3/361/6272492 by guest on 18 May 2024 There is a growing recognition that needs more to be done to ensure that health research is fully mobilized to support improvements in health services and ultimately in outcomes for patients (Oliver et al. 2019). However, in seeking to understand the problem and potential solutions, much of the literature has focused on shortcomings of potential evidence users (such as health care practitioners) and their organizations (Ferlie et al. 2000; Currie and Suhomlinova 2006). This has most commonly been conceptualized as a limited absorptive capacity to use research in health care organizations (Zahra and George 2002). Less attention has been placed on the roles and behaviours of academics and their organizations in supporting or inhibiting the use of research. It might be assumed that, as knowledge producers, research organizations will play a full and active role in supporting the use of research. But, as others have shown, research organizations may have conflicting interests that can obstruct research utilization efforts (Kogan and Henkel 1983; Rip 2001). However, there is a drive for academic researchers to build links with individuals and organizations outside of the academy. In the UK, the influential review by Sir Paul Nurse advocated a more dynamic relationship between organizations that produce and those that use healthcare research (Nurse 2015). This forms part of a wider shift towards more networked and collaborative forms of working for public sector organizations (Kislov 2018) and an appreciation of interdisciplinary and team-based science (Roelofs et al. 2019). The importance of engagement as critical to processes of change can also be seen as part of a longer tradition within social science research (Lewin 1958; Habermas 1987). This tradition has been described by Glerup and Horst (2014) as an integration rationality which conceptualizes knowledge production as a fundamentally collaborative process. Much of this work on supporting the use of research evidence focuses on improving relationships between different actors in the evidence system (Zimmerman 2020). This is often defined as linking with those with a ‘stake’ in the research, or ‘stakeholders’ (Boaz et al. 2018). Who these stakeholders differs—ranging from patients, to policymakers, or more generally potential research users. Stakeholder engagement is considered to have a number of potential benefits including improving research questions, research tools and practices, supporting dissemination and building longer term research-practice partnerships. There is also some evidence to suggest that stakeholder engagement might be a potential mechanism for improving research impact in terms of both healthcare practices and outcomes for patients (Kok et al. 2016; Boaz et al. 2018). The literature often refers to a spectrum of approaches to engagement, suggesting that stakeholders can be engaged in diverse ways that range from providing them with information, to consultation or co-producing research with them (Boaz and Metz 2020). The debate has shifted further, with increased emphasis on the potential contribution and challenges involved in co-producing health services research (Filipe et al. 2017). In particular, there has been a debate about what has been described as the ‘dark side’ of co-production (Oliver et al. 2019; Williams et al. 2020). While the terms co-creation and co-production are now widely used, it is less clear to what extent they signal a change in attitudes and practices (Locock and Boaz 2019). There are a range of programmes in place to support stakeholder engagement in research. For example, in the USA there has been a widespread adoption of research practice partnerships (Coburn and Penuel 2016) and the development of the US Patient-Centered Outcomes Research Institute’s (PCORI) database of articles on stakeholder engagement in research, while in Canada knowledge translation activities have been developed to connect researchers with policy stakeholder communities (Gagliardi et al. 2015). The drive to promote links with stakeholders is captured in most knowledge transfer models. In particular, stakeholder engagement aligns with what Best and Holmes (2010) describe as relational models of knowledge transfer. These models represent a shift from preoccupations with conventional dissemination activities, adding a focus on interactions between people using and producing research, including the development of partnerships and the establishment of networks (Best and Holmes 2010). Capturing these interactions has proved challenging for the field of research evaluation for some time (Molas-Gallart and Tang 2011). There are few empirical studies of stakeholder engagement, particularly taking a prospective approach. In 2013, the UK Medical Research Council (MRC) funded a prospective study of stakeholder engagement in an EU-funded tobacco control research programme (EQUIPT). The EQUIPT programme was funded to adapt, scale and spread a return-on-investment tool to support decision making in tobacco control policy in five European countries. The original tool had been developed in the UK with funding from the National Institute for Clinical Excellence and significant investment from a range of specific stakeholders, including commissioners, service providers, public health directors, local authorities, Smokefree Regional Offices and academics (Pokhrel et al. 2014a). The EQUIPT programme had an explicit theory of change that underlined the importance of engaging stakeholders to develop the tool and promote its impact. The MRC study (SEE Impact) provided an opportunity for using ethnographic methods to study how stakeholders were engaged during the course of the EQUIPT research programme and to compare differences between stakeholder engagement in the different countries. In this article, we address the following question: what can we learn from prospectively studying how academics recognize, conceptualize and operationalize stakeholder engagement over the course of a European research project? Research Evaluation, 2021, Vol. 30, No. 3 on changes in the engagement plans and project activities of the EQUIPT team. 2.1 Interviews 2.2 Observations Six stakeholder events were observed comprising: four events for EQUIPT team members and key stakeholders who formed the project’s Research Advisory Group and two events aimed at dissemination beyond key stakeholders. The number of stakeholders who took part in the six events ranged between 22 and 60. The initial objectives of the EQUIPT project for engaging stakeholders in events were to gain feedback on the use of the ROI tool; gain support for the validation of the ROI tool; and discuss and disseminate findings about the development of the ROI tool. Each of the six stakeholder events was observed by two or three SEE-Impact researchers. The events were held in Maastricht, Brussels (two events), Budapest, London and Zagreb. The first event in Maastricht in February 2014 was a 3-day EQUIPT project team meeting. The second event in Brussels in October 2014 was the EQUIPT project’s first annual team meeting and lasted 2 days. The third event in Budapest in September 2015 was the project’s second annual meeting and also lasted 2 days. The fourth event in London in March 2016 was a half day workshop for stakeholders to give feedback to the EQUIPT team on an earlier, similar UK ROI tool which had been in use in the UK for some time. The fifth event took place in Zagreb in June 2016 and was a 1-day international workshop for potential stakeholders from other European countries beyond the five sample countries with the aim of supporting validation of the ROI tool in lower-income European countries. The final 3-day event in Brussels in October 2016 presented the findings of the study. In addition, six EQUIPT team meetings were observed in order to gain insight into the team’s views and attitudes towards stakeholder engagement, and to learn promptly of any amendments to plans for stakeholder engagement. EQUIPT team meetings were held monthly and took place via teleconference because of the spread of team members across the five sample countries and Croatia as co-ordinator for out of sample countries. Meetings lasted approximately 1.5 h. Detailed field notes were taken at EQUIPT programme events, usually by at least two members of the research team. 2.3 Analysis The three interviewers involved prepared detailed summaries after each semi-structured interview. These summaries covered both the content and setting of the interviews and provided ‘thick descriptions’ (Geertz 1973) of how stakeholders were engaged in EQUIPT. The research team used these descriptions to arrive at a first overview of recurring or deviating themes in the data. The interview transcripts and observations were organized in NVivo (QSR International 2020) and an abductive analysis approach was used to provide the data with codes. This abductive approach combines a theory-informed approach to data analysis with empirical insights from the data (Stoopendaal and Bal 2013; Tavory and Timmermans 2014). In practice, this involved a first round of inductive coding, followed by a second round where these codes were compared to existing theory and codes were added or changed. An example is how we inductively developed our code ‘stakeholder engagement’. In our data, this code increasingly reflected activities that could also be seen as data collection for academic purposes. The literature on stakeholder engagement that we used, and the earlier UK experience developing the original tool, however, mainly referred to stakeholder engagement as a way to create ownership and legitimacy. By comparing the meaning of the code in our data with how it is used in our conceptual literature, we observed a contradiction that we could further explore in our analysis. 3. Findings The presentation of the findings of this study will start with an overview of how the EQUIPT team planned stakeholder engagement, and what their rationale for doing such engagement was. Subsequently, findings are presented sequentially focussing around the four main programme events and combining data from event observations, project documents and interviews with the project team and key stakeholders. 3.1 Stakeholder engagement planning and rationale The aim of EQUIPT was to develop country-specific tools to support decision makers (including local policy makers and those procuring public health interventions) in accessing predictions of likely returns on investment arising from funding different tobacco control interventions (Pokhrel et al. 2014b). EQUIPT set out with a clear work plan to engage with stakeholders from the beginning and throughout the programme. A wide range of terms were used by the team to describe work with potential stakeholders’ including stakeholder engagement, co-creation and co-production. The study protocol used the language of ‘co-creation’ to signal their intent to work closely with stakeholders on the adaption, scale and spread of the returnon-investment tool to other European countries (Pokhrel et al. 2014b). Within the study design, there were two elements with an explicit focus on stakeholder engagement: in the so-called ‘working space’ where the return-on-investment tool was to be developed (where a process of co-creation with stakeholders was envisaged) and in the so-called ‘transfer space’ where stakeholder engagement was considered to be integral to the process of disseminating the return-on-investment tool. The EQUIPT funding agreement part B document states that the following stakeholder groups would be targeted: ‘(1) National and European stakeholders consisting of policymakers, academics, health authorities, insurance companies, advocacy groups, ministry of finance, national committees, clinicians and health technology assessment (HTA) professionals—the outcomes of engagement with Target Group 1 will be used to obtain an optimum assessment of preconditions for usability of the final Downloaded from https://academic.oup.com/rev/article/30/3/361/6272492 by guest on 18 May 2024 Fifty interviews were conducted with stakeholders (n ¼ 45) and with members of the project research team (n ¼ 5). The stakeholder interviews comprised six in Germany, eight in Hungary, thirteen in the Netherlands, nine in Spain and nine in the UK. Interviews took place between April 2015 and September 2016. In Germany, Hungary, the Netherlands and Spain, interviews were conducted face-to-face. In the UK, eight interviews were conducted over the telephone and one was face-to-face. Interviews were digitally recorded, translated into English where relevant, and transcribed. Questions were open-ended and investigated the circumstances around stakeholders’ awareness of and involvement in EQUIPT, expectations of involvement in the project, the type and level of interaction with the EQUIPT team, benefits gained through working with EQUIPT, the perceived influence of stakeholder engagement on the project, and barriers to effective engagement. 363 364 ‘It is about a way of working, participation and coalition building around a piece of work that creates that acceptability, that willingness to create usability that had been so important in the previous work. . .Those who have been engaged are much more likely to use. Jack Smith [An attendee at the final event] was one of the original stakeholders in the [UK] ROI project and is now still engaging with us from a policy level perspective’ Angela, UK regional smoking policymaker Jack had been engaged as a policy stakeholder in the development of the previous ROI tool. His role as a stakeholder had changed over time, but his relationship with the team and his interest in and understanding of the ROI product meant that now, in a more senior and influential role, he could be a powerful ally in supporting the use of the new EQUIPT tool in UK smoking cessation policy. This understanding of the dual roles of stakeholder engagement in improving the quality of the tool and supporting potential future use seemed to be shared throughout the team and at different levels of seniority from the outset. One of the more junior researchers articulated it as follows: ‘The idea is that we should involve stakeholders at an early stage anyway, probably because they then feel that they can also provide input in the development of the tool and that ultimately leads to more easy behavioural change. Um and that the tool is better adapted for them.’ Peter, EQUIPT researcher This view was echoed by more senior colleagues: ‘So basically everything is used for, everything is, everything has the idea that, by involving stakeholders, you can make the model more tailored to their wishes and their ideas, making it better and more used, so to speak.’ Ana, Theme Lead, EQUIPT This quotation demonstrates the different theoretical frameworks individual members of the EQUIPT team were drawing on. In this case, individual behaviour change models were dominant in the thinking about stakeholder engagement of both the individual researcher and his colleagues in the work package, several of whom were psychologists by background. 3.2 How stakeholder engagement in the project evolved 3.2.1 Maastricht—February, 2014 At the first annual project meeting, the work package leading on stakeholder engagement presented a strategy for categorizing key stakeholders into five groups. These groups were: (1) decision makers, (2) purchasers of services or pharmaceutical products, (3) professionals service deliverers, (4) evidence generators (e.g. researchers) whose work informs policy, procurement, or delivery of services and (5) advocates of health promotion. In addition, the work package presented a shared methodology for initial engagement with stakeholders through face-to-face interviews. There was a very positive reaction from across the team to work done to conceptualize stakeholders into different groups and to develop a detailed interview questionnaire to gather stakeholder views at the outset of the study. Some of the team voiced concern over whether it would be possible to engage with every stakeholder category across the different countries. They argued that some groups would be less prevalent, or generally less inclined to participate in scientific projects. The Maastricht meeting was also intended to explain and pilot the stakeholder questionnaire among the EQUIPT team members. A junior researcher, who was hired to co-develop the questionnaire and coordinate the first work package, would later explain the objective of using the questionnaire as follows: “. . . the purpose of the questionnaire is to improve the tool, so that we are provided with input on how we can develop the tool in such a way that it aligns as much as possible with the stakeholders. So, we look at what evidence types they are interested in, but also perhaps which usability, um, characteristics they find necessary. But I really combined this with scientific research that we find interesting, namely we have integrated that with the IChange model. . . So, we have the objective for EQUIPT and an objective to conduct our own research and we integrated that in a questionnaire. Um, there is overlap, But then you can understand better why we used a questionnaire in the interview, because we, because they, because the stakeholders can score on all items of the I-Change model. So that we can do research and can look at what we can improve about the tool. So, they were not Downloaded from https://academic.oup.com/rev/article/30/3/361/6272492 by guest on 18 May 2024 ROI tool (Task 4); and (2) Experts on smoking cessation and HTA—the outcomes of engagement with Target Group 2 will be used to obtain an optimum assessment of the parameters to be included in the final ROI tool, taking into account the variability of smoking cessation/prevention methods used between different countries.’ (EQUIPT Description of Work Part B) A work package, with a detailed programme of engagement activities, was dedicated to working with stakeholders. There were also stakeholder engagement activities planned within other work packages to support tool development and dissemination. In total, the proposed stakeholder engagement activity detailed in the description of work for the study consisted of: a stakeholder survey across five countries (EQUIPT Description of Work Part A, p3 of 23, last para), 10 consensus workshops within each country and two further consensus workshops across all countries (EQUIPT Description of Work Part A, p4 of 23, last para.). Piloting of the country-specific ROI tools involving all stakeholders engaged in previous activities (EQUIPT Description of Work Part A, p7 of 23, last 3 paras); Consultation with local researchers and policy makers in the out of sample Central and Eastern European countries regarding collection of local data. (EQUIPT Description of Work Part A, p10 of 23, 3rd para); a workshop of UK regional and national stakeholders (EQUIPT Description of Work Part A, p12 of 23, 2nd para.); an international workshop of stakeholders in non-sample Central & Eastern Europe countries, (EQUIPT Description of Work Part A, p3 of 23, 4th para); A project website needs-assessment conducted via an online survey organized by country leads. (EQUIPT Description of Work Part A, p14 of 23, 1st para); Invitations to stakeholders to attend selected project meetings as appropriate (EQUIPT Description of Work Part A, p15 of 23, 3rd para); Network Building by collaborating with key networks and health organizations. (EQUIPT Description of Work Part A, p15 of 23, 4th para). This commitment to stakeholder engagement had roots in the prior work of a number of the co-investigators who had had a positive experience of stakeholder engagement as part of the development of a previous project. The funder of the original return on investment tool study in the UK supported a high level of stakeholder engagement in the work that it funded (Pokhrel et al. 2014a). At the final event, one of the EQUIPT team who had been engaged in the prior work in the UK and had a policy role, reflected on the potential for stakeholder engagement to build long term relationships, support and potential for use: Research Evaluation, 2021, Vol. 30, No. 3 Research Evaluation, 2021, Vol. 30, No. 3 really real interviews, it was more really very, it were very structured interviews in which they really just had to fill out a questionnaire.” Peter, EQUIPT researcher Matthew [EQUIPT stakeholder]: I believe that he had a, a, uh, list with questions that he followed quite strictly, I think. And he had, yes, he had a computer with him, on which he showed things. A laptop or something. Interviewer: Yes, he probably showed a video? Matthew [EQUIPT stakeholder]: Yes! Yes! And that was surrounded by some sort of standardised story, by him, and that is what he then did entirely: telling that standard story. And, and, uh, asking questions before, yes. Watching something, asking questions afterwards and then at the end there was this sort of standard question, like: do you have other comments? That is when I mentioned several things. Things that, for me I think, were most important. And I have no notion whatsoever as to what happened with those things. At this point, the idea that stakeholders would be beginning to see themselves as partners in the research process with some ownership of the tool seems to be slipping away, as Matthew describes an experience of participating in more of a one-way data collection exercise. 3.2.2 Brussels—October 2014 By the second annual project meeting in Brussels, a number of challenges to stakeholder engagement were emerging. In particular, the time required to gather feedback directly from stakeholders was proving difficult to reconcile with the needs of the modellers (working on the new return on investment tool), the demands of the technical tool adaptation process and development process. This was further compounded by the decision by the funder to require all grant holders to adhere to their original timeline (with end dates remaining set regardless of project delays) (Boaz et al. 2018). The need to deliver the adaptation of the tool on time led to a rethinking about the intensity and method for stakeholder engagement. After much discussion, the large number of face-to-face meetings planned with stakeholders were replaced by recorded SKYPE calls with stakeholders focused on testing the adapted tool. In addition, the planned ‘consensus workshop’ was reframed into a usability test of the model. Initially, the workshop aimed at arriving at consensus among stakeholders regarding the most important smoking-related diseases to include in the model. At this stage, however, the modellers seemed concerned that this step would further lag the already delayed development of the tool. ‘At the end of the first project day, it seems that the modellers are constantly asked to change their variables and input data. Although they articulate their concerns quite modestly, their faces express unease. Discussion is constantly focussed on what is in or outside the model’ exert from SEE-Impact researcher field notes One of the senior researchers in the project suggested to focus on usability of the model instead. Using terms as ‘back-up plans’ and ‘thinking out of the box’, he hinted at a digital approach where stakeholders could use the model whilst researchers could gather data about the usability of the model—thus noting a shift towards a more dissemination focussed engagement strategy. 3.2.3 Budapest—September 2015 By the third annual meeting in Budapest, a further shift had occurred which seemed less driven by the technical elements of tool development and the requirements of the modellers. At this stage, the EQUIPT research team seemed to be describing a pull towards generating knowledge suitable for publication. The researchers reported on a decision to conduct a survey of stakeholders to gather feedback on the tool. This validation exercise was conducted in a more conventionally scientific format. While the researchers discussed the results in terms of their utility for tool development, they also emphasized the potential for generating academic publications based on the results. As one of the SEE-Impact research team observed in her notes: ‘It is possible to see how [research with stakeholders rather than stakeholder engagement] continues to gain insights for the development of the tool. In fact it may gain more detailed feedback through the observations of individuals as they use the tool. However, is it likely to build ownership of the tool in the same way as the planned face to face stakeholder engagement exercises?’ exert from SEE-Impact researcher field notes At this point, the dual goals of stakeholder engagement in EQUIPT (improving the tool and promoting use of the tool) seemed to uncouple and shift. While the importance of stakeholder views in shaping the tool remained in the foreground, the opportunity to recast stakeholder engagement as a research activity for academic publication purposes proved attractive to some of the team members. At this stage in the process, the second goal of stakeholder engagement (to build relationships and ownership of the tool in order to promote use) was mainly supported by some of the country teams, who still felt this was crucial for the success of the tool. In particular, the Spanish and Hungarian team seemed to be more committed to a more personalized approach to stakeholder engagement, as reflected in the words of a Spanish EQUIPT researcher: “The first sentence was: ‘This questionnaire is to test the users’. Maybe if you are a stakeholder you will think: ‘okay, what are they asking me, what will they ask me?’. If [the interviewers] are stating that in the very first thing in the questionnaire; it makes [the stakeholders] suspect that it will be an exam. (. . .) So, I told [the Dutch questionnaire developers]: keep that in mind, that the interview, it is not an exam. Don’t see it like an exam. But I think the Hungarians said something related to that as well” Lucia, EQUIPT researcher A Hungarian EQUIPT member would later explain that some country teams: ‘(. . .) may have a different perspective on [engagement] and a different information need. For them the Downloaded from https://academic.oup.com/rev/article/30/3/361/6272492 by guest on 18 May 2024 The questionnaire itself was composed of 19 structured questions, mostly requiring Likert-type answers and including several sub-questions. The information sheet of the questionnaire explained that the interview ‘(. . .) should last approximately 40 min’ and— with the respondent’s verbal agreement—would be audio recorded ‘to save time’. Most of the survey interviews with EQUIPT stakeholders were conducted between January and July 2014. Shortly after, the SEE-Impact team approached several of these stakeholders for a semi-structured interview on their experiences being engaged with EQUIPT. Most stakeholders explained that they could not recall the exact content of the interview, nor whether they were satisfied with it. Stakeholders like Matthew expressed some confusion about what and how they were being engaged in the EQUIPT study: 365 366 3.2.4 Brussels—September 2016 The EQUIPT project was concluded with an end-of-project meeting in Brussels. The meeting covered 3 days, of which one was dedicated to discussion with the research advisory group, another day was focussed on presenting the tool to external guests, and the final day was an internal meeting for the research team. At the end of the first day, the tool itself had yet to be presented: ‘Some researchers in the meeting seem to be rather distant from the non-academic tobacco control practice. The entire meeting and I have not seen the model itself yet, whilst all the technical aspects have been exhibited. It feels a little like selling a vehicle, but then only showing the engine bay and obfuscating the exterior.’ exert from SEE-Impact researcher field notes The second day was opened by a former Minister of Health from Austria. Afterwards, EQUIPT team would continue to present three themes: (1) stakeholder engagement in EQUIPT, (2) EQUIPT and decision making and (3) transferability of the tool. The first theme was meant to share lessons derived from the various stakeholder engagement activities. A presentation from Ana, a senior EQUIPT researcher, emphasized that stakeholder engagement ‘takes a lot of effort, especially if you have to build a model from scratch’. They would continue to explain that the team ‘also wanted to maintain [their] scientific integrity’—which sometimes led to exclusion of stakeholders or their inputs. During lunch, one of the observers of the SEE-Impact study asked a lead modeller whether the tool itself would be presented during the meeting. The modeller explained that such a presentation was not scheduled and that, given uncertainty over the stability of the tool, they could risk disappointing the stakeholders. He also confessed that they had not actually considered a live presentation. After a short deliberation among the EQUIPT team, they decided to showcase the tool shortly after lunch. At the team meeting following the final stakeholder event in Brussels, members of the team reflected on the misfit between the style and content of the event (with a series of academic presentations from a podium) and what they felt on reflection the audience might have appreciated (an opportunity for stakeholders to directly engage with the new ROI tool). The team had spent sufficient time discussing the importance of stakeholder involvement throughout the project to see that the event they had organized was more suited to an academic audience than to the assembled group of potential users of the tool. One of the team commented that ‘I don’t think we (the team and the audience) were speaking the same language’ she talked about the lack of a ‘real connection’ with stakeholders. Comparisons were drawn with a previous stakeholder event in Zagreb (led by the Hungarian team) which had provided plenty of opportunities for interactions between the team and stakeholders and was considered by many in the team to have set a higher bar in terms of expectations of stakeholder engagement in the project. One of the co-applicants (Ana, theme lead) reflected on the potential tensions for the team between close working with stakeholders and the importance of maintaining academic integrity. There was considerable variation within the EQUIPT team regarding the importance of stakeholder engagement. While stakeholder feedback was consistently welcomed, valued and responded to by members of the team, there was less consensus about its wider potential value in relation to use and impact. In some of the EQUIPT countries, the stakeholder engagement was directly associated with an idea to increase the tool’s use. In these countries, the responsible EQUIPT team maintained friendly contact with actors that could be seen as potential users of the tool. Other countries adopted a more generic understanding of potential users, such as ‘decision makers’ or ‘researchers’. Some EQUIPT research team members appeared to place greater significance on stakeholder engagement than other team members. They talked about the importance of more ‘hands on’ and ‘practical’ input from stakeholders and viewed the stakeholder events as being too static and research focussed. In their language, they placed a value on fully engaging and making a ‘real connection’ between stakeholders and the EQUIPT team. This perspective aligned with the underpinning theory of change which outlined a more bottomup approach to its stakeholder engagement, ‘working with people who are going to be making decisions in the future (and with current stakeholders) to work together to convince politicians’ [researcher observation notes]. However, for some, this may have been to do with practical issues such as awareness of time restraints around the project and the need to deliver the tool on time. Some members of the team prioritized the academic elements of the project (e.g. the completion of a PhD by a team member and academic publications), often directly at the expense of stakeholder engagement activities. Furthermore, when this tension came to a head at a meeting of the EQUIPT project team and its advisors, the promise of turning stakeholder engagement activity into research data and academic outputs gained support from the partnership as a whole. The prospective of applying for follow on EU Horizon 2020 funding potentially provided a further incentive to focus on outputs. The ‘irresistible pull’ of academic norms proved too strong to resist, despite a considerable interest and commitment to stakeholder engagement in the programme. Interview participant: I don’t know, um, the stakeholders have different ideas as well. Probably some say, then you know, then you have, then you know, more difficult, then it is more difficult to determine the direction of what you are going to investigate, I think. Interviewer: Yes. Interview participant: And maybe we think something, as a scientist, is very important and then they say that it is absolutely not important, and then if they have a very large part in the process, Downloaded from https://academic.oup.com/rev/article/30/3/361/6272492 by guest on 18 May 2024 documentation, the screenshots, might be very useful for the research purpose.’ [Vilmos, EQUIPT researcher]. When he was asked to explain how this approach would affect the overall engagement of stakeholders he said: ‘If you want to distinguish yourself, and if you want to build a more personal relation to them - especially, if you want them to later use the tool - I think a personal meeting would have been a better option.’ The shift towards a more scientific stakeholder engagement approach was not the case for all the work presented in Budapest, and the perceived needs of stakeholders were regularly mentioned by members of the team throughout the meeting. The work packages presented by non-academic collaborators continued to emphasize the importance of bringing people together: The team looking at the transferability of the tool to other European countries presented their plans for engaging with stakeholders in a face-to-face event in Zagreb to explore issues of transferability. Besides, the UK team was looking to convene a meeting of stakeholders who had used the previous ROI tool to identify learning for the implementation of the new EQUIPT tool. Research Evaluation, 2021, Vol. 30, No. 3 Research Evaluation, 2021, Vol. 30, No. 3 then you can no longer, then you can no longer do your own thing.’ Peter, EQUIPT researcher Here, Peter, one of EQUIPT project team reflects on some of the difficulties that stakeholders’ feedback presented for the technical tool development work and, to the threat to his academic autonomy in stakeholders affecting his ability to ‘determine the direction’ of his work. Ana reflected on how different stakeholder engagement would have felt with the stakeholders sitting ‘at the table’ rather than completing a survey. Interviewer: Yes Interview participant: Um, while I think if someone really sat at the table where those decisions are made, it might have been something else. [Ana, theme lead, EQUIPT] 4. Discussion The collaborative work in the EQUIPT project was identified as a key feature of the study design with pre-designated co-creation spaces within the work programme—the so called ‘working space’ and ‘transfer space.’ Over the course of the study, however, the tool development work and stakeholder engagement activities decoupled and ceased to occupy a shared space. The impetus for the decoupling seemed to come simultaneously from two directions and was facilitated by the organization of the activities in different engagement and production work packages. Despite the plans for co-creation in the working space of the programme, the technical work on the modelling for the new tool increasingly took place in parallel to what, by then, was more conventional consultation. Modelling and tool development increasingly occurred in the sort of ‘secluded’ space described by Callon et al. (2009), where there is a technical job of work to be done and outside influence can often be characterized as uncertain, unpredictable and lacking timeliness. The real-world challenges presented by delays in commencing the study and the time required to set up face-to-face stakeholder engagement had significant implications for the modellers working to a tight timetable to develop new versions of the tool for prototype testing in the different countries. In the course of the project, most of the planned ‘co-production’ activities subtly transformed to consultation, and eventually, for some of the activity, to research participation where stakeholders completed surveys generating data that was subject to detailed analysis and written up for publication in peer-reviewed journals. A first step, responding to time pressures in project delivery, involved replacing the planned face-to-face stakeholder workshops with online one-toone consultations, where developments in technology made it possible to make both audio and visual recordings of stakeholders testing the prototype tools. While this process generated detailed data on the different elements of the prototype tool from stakeholders, it did not do so in close collaboration with the modellers. While the project team seemed to form a successful interdisciplinary collaboration of academics (Roelofs et al. 2019), the eventual collaborative research practice did not result in similar opportunities for building engagement with and ownership of the tool amongst a wider group of stakeholders across the different countries. One of the EQUIPT studies shows that Hungarian and Spanish stakeholders ‘wanted to use the tool basically as soon as possible’ whereas ‘Dutch and German interviewees were least interested’ (Vokó et al. 2016). A previous SEE-Impact study has described how this difference relates to the more personalized approach by the Hungarian and Spanish project teams, where the notion of ‘stakeholders’ was commonly translated into concrete actors and positions (Borst et al. 2019). The study also surfaced a more fundamental challenge to close working with stakeholders in knowledge production and use. Mary Henkel’s work (2005) on academic identity and autonomy in changing policy environments explored how academics respond to the promise and challenges of working closely with stakeholders outside of the academy. It is also echoed in the findings of Timotijevic et al. (2013) study of stakeholder involvement in scientific decision making where they observed stakeholder engagement being used to confirm the authority of science over the facts as opposed to any evidence of a reframing of scientific practice. As Morgan et al. (2011) observe, existing university policies and practices support particular models of knowledge production. As a consequence, more applied, collaborative approaches to research can end up left to one side as the dominant model of academic knowledge production (driven by the science and not primarily concerned with applicability and use of research findings) asserts its authority. The value of stakeholder engagement was articulated clearly and consistently by the EQUIPT project team throughout. This reflects what Goffman (1963) would characterize as a shared performance front of stage. Furthermore, the substantive content of the work aligned with their values in terms of promoting the importance of supporting a more evidence-based approach to making return on investment decisions in tobacco control. Several members of the EQUIPT team highlighted their prior positive experience of stakeholder engagement in developing the UK ROI tool. Where this commitment started to unravel was ‘backstage’ where the more intensive engagement and co-production elements started to ‘rub up against’ the priorities and ways of working of academia. Kislov et al. (2017) and Lozeau et al. (2002) note the impact of similar ‘compatibility gaps’ between new practices and the cultural, structural and political characteristics of the system in their studies of service improvement. While participants in the study had fully internalized the importance of academic writing and grant writing as an integral part of their role, stakeholder engagement, and in particular the planned co-production activities remained vulnerable to internal and external pressures. While there were many differences within the team, there was concordance on the importance of publications. Despite the substantial stakeholder engagement built into the study throughout, the importance of academic publications ‘trumped’ stakeholder engagement at every turn. There was an exuberant moment when the team as a whole saw the opportunity to publish the outcomes of their stakeholder engagement work in a peer-reviewed journal. The vulnerability of working with stakeholders has particular implications for the currently promotion of co-production of knowledge in health services research. Our tendency to see involving stakeholders as a benign ‘add on’ that will enhance the quality of our research misses the underlying epistemological challenge presented by stakeholder engagement and in particular by co-production as described by Jasanoff (2004): Downloaded from https://academic.oup.com/rev/article/30/3/361/6272492 by guest on 18 May 2024 Interview participant: Yes, well, I mean, what happened here [in EQUIPT]: the interviewer asks the stakeholders something, the interviewers summarize that, report it to the researchers, the researchers say ‘yes we do’ or very often ‘we do not for those and those reasons’, and then after a while we start asking the stakeholders again. You know, and that was it. 367 368 ‘Co-production can therefore be seen as a critique of the realist ideology that persistently separates the domains of nature, facts, objectivity, reason and policy from those of culture, values, subjectivity, emotion and politics.’ (Jasanoff 2004: 3) Acknowledgement The authors would like to thank the EQUIPT consortium (http:// equipt.eu) for participating in our study. In particular, we are grateful to Subhash Pokhrel and Teresa Jones who acted as a bridge between the two studies. We very much appreciated the constructive comments of our colleague Steve Hanney and also of the two peer reviewers. Finally, we would like to thank the United Kingdom Medical Research Council for funding this work. Funding UK Medical Research Council (MR/L011123/1). Conflict of interest statement. None declared. References Best, A. and Holmes, B. 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Disease-Induced Alterations in Brain Drug Transporters in Animal Models of Alzheimer’s Disease Kati-Sisko Vellonen, Jouni Ihalainen, Marie-Christine Boucau, Fabien Gosselet, Théo Picardat, Mikko Gynther, Katja Kanninen, Anthony White, Tarja Malm, Jari Koistinaho, et al. To cite this version: Kati-Sisko Vellonen, Jouni Ihalainen, Marie-Christine Boucau, Fabien Gosselet, Théo Picardat, et al.. Disease-Induced Alterations in Brain Drug Transporters in Animal Models of Alzheimer’s Disease. Pharmaceutical Research, 2017, 34 (12), pp.2652-2662. �10.1007/s11095-017-2263-7�. �hal-02506588� HAL Id: hal-02506588 https://univ-artois.hal.science/hal-02506588 Submitted on 23 Jul 2021 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. 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. Distributed under a Creative Commons Attribution 4.0 International License Pharm Res (2017) 34:2652–2662 DOI 10.1007/s11095-017-2263-7 RESEARCH PAPER Disease-Induced Alterations in Brain Drug Transporters in Animal Models of Alzheimer’s Disease Theme: Drug Discovery, Development and Delivery in Alzheimer’s Disease Guest Editor: Davide Brambilla Kati-Sisko Vellonen 1 & Jouni Ihalainen 1 & Marie-Christine Boucau 2 & Fabien Gosselet 2 & Théo Picardat 1 & Mikko Gynther 1 & Katja M. Kanninen 3 & Anthony R. White 4 & Tarja Malm 3 & Jari Koistinaho 3 & Markus M. Forsberg 1 & Marika Ruponen 1 Received: 20 June 2017 / Accepted: 12 September 2017 / Published online: 26 September 2017 # Springer Science+Business Media, LLC 2017 ABSTRACT Purpose Alzheimer’s disease (AD) may disturb functions of the blood-brain barrier and change the disposition of drugs to the brain. This study assessed the disease-induced changes in drug transporters in the brain capillaries of transgenic AD mice. Methods Eighteen drug transporters and four tight junctionassociated proteins were analyzed by RT-qPCR in cortex, hippocampus and cerebellum tissue samples of 12–16month-old APdE9, Tg2576 and APP/PS1 transgenic mice and their healthy age-matched controls. In addition, microvessel fractions enriched from 1-3-month-old APdE9 mice were analyzed using RT-qPCR and Western blotting. Brain transport of methotrexate in APdE9 mice was assessed by in vivo microdialysis. Results The expression profiles of studied genes were similar in brain tissues of AD and control mice. Instead, in the microvessel fraction in APdE9 mice, >2-fold alterations were detected in the expressions of 11 genes but none at the protein level. In control mice strains, >5-fold changes between different brain regions were identified for Slc15a2, Slc22a3 and Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11095-017-2263-7) contains supplementary material, which is available to authorized users. * Kati-Sisko Vellonen Kati-Sisko.Vellonen@uef.fi 1 School of Pharmacy, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, FI, Finland 2 Laboratoire de la barrière hémato-encéphalique (LBHE), University Artois, Lens, France 3 A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland 4 QIMR Berghofer Medical Research Institute, Herston, QLD, Australia occludin. Methotrexate distribution into hippocampus of APdE9 mice was faster than in controls. Conclusions The expression profile of mice carrying presenilin and amyloid precursor protein mutations is comparable to controls, but clear regional differences exist in the expression of drug transporters in brain. KEY WORDS APdE9 . blood-brain barrier . brain disposition . brain microdialysis . CNS exposure . pharmacokinetics ABBREVIATIONS AD amyloid-β BBB CB CX HC wt Alzheimer’s disease Aβ Blood-brain barrier Cerebellum Cortex Hippocampus Wild type INTRODUCTION Alzheimer’s disease (AD) is the most common form of dementia; it is associated with accumulation of senile plaques and neurofibrillary tangles in the brain (1). The extracellular senile plaques are mainly formed by aggregating amyloid-β (Aβ) peptides in the brain parenchyma, whereas hyperphosphorylated and misfolded tau proteins are the main constituent of the intraneuronal neurofibrillary tangles. The distribution of drug molecules from the systemic circulation to the brain is controlled and limited by the blood-brain barrier (BBB). The BBB is formed by capillary endothelial cells and supported by the end feet of astrocytes and pericytes around brain capillaries. Dysfunction of the BBB has been Characterization of Drug Transporters in AD Models associated with neurodegeneration, although the importance of BBB alterations solely attributable to AD, without the presence of additional vascular diseases, is still unclear (2). A dysfunctional BBB may alter the access of AD drugs to their site of action; it may either increase the drug exposure to the brain, potentially causing unwanted effects, or decrease drug access and lead to an insufficient response. AD patients are generally over 65 years old, and in addition to AD drugs, they often have medication for several other chronic diseases. Therefore, BBB dysfunction can also either increase or decrease the brain distribution of other concomitant medications and possibly increase their adverse effects on the central nervous system. The BBB is enriched with several transporter proteins belonging to the ATP binding cassette (ABC) and solute carrier (SLC) gene families; these are responsible for ensuring the supply of dietary components into the brain and the disposal of metabolic products from the brain, as well as preventing noxious agents accessing the brain from the circulation (3). Transporters have also a significant role in the pharmacokinetics of many drugs. For instance, L-dopa, which is used to treat Parkinson’s disease, crosses the BBB via the influx carrier L-type amino acid transporter (LAT1). Furthermore, the efflux transporter Multidrug resistance protein 1 (MDR1, Pglycoprotein) is known to diminish the central access of various drug molecules. In addition, high levels of efflux transporters including Breast cancer resistance protein (BCRP, ABCG2) and Multidrug resistance-associated protein 4 (MRP4, ABCC4) are expressed by the endothelial cells in the BBB (4). Other transporters, relevant also as drug carriers, such as MRP proteins (MRP1, MRP5), monocarboxylate (MCT1), nucleoside (ENT1) and organic cation (OCT3) and anion (OAT3) transporters, organic anion transporting polypeptides (OATPs) and multidrug and toxin extrusion protein 1 (MATE1) have been localized in the endothelial cells of human and mouse brain capillaries (3–7). The influence of AD on the expression and functionality of some BBB drug transporters has been studied both in postmortem AD patient samples and animal models of AD. MDR1 has been the most widely studied efflux protein in AD and in addition to a wide variety of drug molecules it has been shown to also transport Aβ (8). Downregulation of MDR1 expression in brain capillaries in close proximity to Aβ deposits of AD patients has been observed by several groups (9–13). In addition to expression, a positron emission tomography study investigated MDR1 function in the BBB of AD patients with MDR1 substrate (R)-[11C]verapamil and found evidence of decreased efflux function (14). The findings related to the impact of AD on BCRP expression have revealed minor and also partly conflicting alterations (10,12,13,15) and a slight upregulation of MRP4 expression at both RNA and protein levels has been detected in the hippocampus of AD patients (10). There are even fewer studies examining diseaseinduced changes in drug transporter expression in AD mouse 2653 models. With respect to Mdr1, both up- and down-regulation have been reported, whereas increase in expression levels of Bcrp have been detected (8,16,17). However, there are many transporters whose expression and functionality in AD remain obscure i.e. no comprehensive comparative data is available. Transgenic mouse models of AD, which have mutations in amyloid precursor protein (APP), such as Tg2576, or in both APP and presenilin (PSEN1) genes, such as APdE9 and APP/ PS1 mouse models, can provide useful information on AD pathogenesis and disease mechanisms. APdE9 mice exhibit an increase in the parenchymal Aβ load, with the appearance of Aβ plaques starting from the age of four months; APP/PS1 mice develop Aß deposits around 9 months of age, and Tg2576 mice around 10 months of age. In addition, signs of gliosis can be seen concurrently. Disease models are important tools for drug development. For example, the pharmacokinetic properties of novel AD drug candidates can be studied in models having similar disease-induced alterations as AD patients. Before these AD models can be better exploited in preclinical drug development, it would be important to acquire a more detailed characterization of the drug transporters. This study aimed to characterize the expression of drug transporters and tight junction-associated proteins in three mouse models of AD, the APdE9, APP/PS1 and Tg2576 mice, and to investigate the regional expression differences in the brain. In order to assess the effect of AD pathology on drug transport into the brain in vivo, we applied a pharmacokinetic microdialysis method to measure the unbound extracellular drug levels in the hippocampus after systemic administration of methotrexate. MATERIALS AND METHODS Animals All procedures were made in compliance with the European Communities Council Directive (86/609/EEC) and Principles of laboratory Animal Care, and studies were approved by the National Animal Experiment Board. The animals were kept in a controlled environment with 12 h light/ dark cycle and food and water freely available. Brain tissue samples were collected from male APP/PS1dE9 (APdE9) mice (C57BL/6 J strain) (18) at the age of 16 months (n = 3– 4), female APP/PS1 mice (C57BL/6 J strain) (19) at the age of 12–15 months (n = 3–4) and female Tg2576 mice (C57BL/ 6JxSJL strain) (20) at the age of 12–15 months (n = 4). All mice groups had age-matched wildtype (wt) controls (n = 3–5). During sample collection, the mice were anesthetized with 250 mg/kg Avertin and perfused 3 min transcardially with heparinized (2500 IU/l) saline at a flow rate of 20 ml/min. The cortical (CX), hippocampal (HC) and cerebellar (CB) samples were dissected and snap frozen in liquid nitrogen. 2654 Aβ accumulates mainly in CX and HC, whereas CB remains devoid of Ab deposits and was therefore considered as unaffected control tissue. Mechanical Isolation of Brain Capillaries The microvessel enriched fraction was isolated from 5–12 weeks old female APdE9 mice as previously described by Coisne et al. (21). Briefly, ten cortices, from APdE9 or wt mice were isolated by removing cerebellum and brain white matter. Outer vessels and meninges were also removed. Preparations were pooled and ground using a Dounce homogenizer (first with the loose clearance pestle: 25–80 μm, then with the tight clearance pestle: 5–25 μm) in HBSS 1x containing 10 mM HEPES and 0.1% BSA. The resulting homogenate was mixed with 30% dextran (v/v, molecular weight 100,000–200,000) in HBSS 1x supplemented with 10 mM HEPES and 0.1% BSA. This suspension was centrifuged at 3000 g for 25 min at 4°C. The neural component and the dextran layer were discarded and the pellet containing the vascular component was washed twice in cold PBS CMF and then separated into two equal parts by centrifugation at 1000 g for 7 min at 4°C. The resulting pellets were resuspended either in RLT lysis buffer for RNA extraction or in RIPA lysis buffer for protein extraction. Quantitative RT-PCR RNA expression of drug transporter and tight junctionassociated genes was studied in brain tissues of 12–16 months old mice modelling AD and their healthy age-matched controls. Total RNA was extracted from HC, CX and CB tissue samples with TRI Reagent (Sigma). From microvessel samples, the RNA was extracted by RNeasy Mini Kit (Qiagen). After RNA extraction, the procedure was similar for both tissue and microvessel samples. Possible DNA residues were removed with DNA-free™ kit (Ambion). RNA concentration was measured with Nanodrop 1000 (Thermo Fisher Scientific) or NanoVue (GE Healthcare) spectrophotometer and 500 ng (brain tissues) or 200 ng (microvessels) of RNA was reverse-transcribed using random primers and M-MuLV enzyme (Fermentas, Hanover, MD). In the quantitative realtime PCR, the ABI Prism 7500 instrument with TaqMan® Gene expression assays (Supplementary Table I) or SYBR Green chemistry and primers designed for each gene (Supplementary Table II) were used. Normalized expression was calculated with the QGene application (22) using betaactin as the reference gene. In the microvessel enriched fraction, a 2-fold difference was used as a threshold value and when a > 2-fold difference was determined with the first batch, the results were confirmed with a second batch. Efficiency was calculated for each primer pair and a melting curve analysis was performed after amplification cycles, in order to check the specificity/purity of each amplification. Kati-Sisko et al. Western Blotting After the capillary isolation procedure, the pellet was washed twice with cold PBS-CMF and then homogenized (polytron homogeniser: 2 times for 15 s at 11000 r/min on ice) in cold RIPA lysis buffer (Millipore, containing 0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40 and 10 mM EDTA, supplemented with phosphatase and protease inhibitor cocktails (Sigma-Aldrich)). The protein concentration in each sample was determined by the method of Bradford (Bio-Rad). Then, 5–40 μg of proteins were electrophoresed on 4–15% Criterion XT sodium dodecyl sulphate (SDS)-polyacrylamide gel (P-gp/Abcb1, Rage, Lrp1) or 4– 20% Mini-Protean TGX Gel (Mrp4) (Bio-Rad) and subsequently electrotransferred to nitrocellulose membranes. Unspecific binding was blocked with 5% skimmed milk in TBS, 0.1% Tween 20 and then incubated with primary antibody using following dilutions: Rage (Abcam) 1:2000, P-gp/ Abcb1 (C219) (Genetex) 1:400, Lrp (5A6) (Santa Cruz) 1:200, Mrp4 (M41–10) (Santa Cruz) 1:200, β-actin (AC-15) (SigmaAldrich) 1:20,000, α-tubulin (B-5-1-2) (Sigma-Aldrich) 1:8000 and β-tubulin (9F3) (Cell Signaling) 1:1000. Immunoreactivity was detected with HRP conjugated secondary antibodies (Goat anti-rat IgG (Santa Cruz) (1: 10,000), Goat anti-rabbit IgG (Dako) (1:2000) and Goat anti-mouse IgG (1:2000 for Dako and 1:7500 for Santa Cruz) and enhanced chemiluminescence (ECL) system. Pharmacokinetic In Vivo Microdialysis Microdialysis experiments were performed with 16–18 months old male APdE9 mice. To implant the guide cannulae (Brainlink B.V., the Netherlands) 2–3 days before the microdialysis experiment, the mice were anesthetized with 1–2% isoflurane in 70% N2O and 30% O2 (flow 300 ml/min, induction 5% isoflurane). Lidocaine (10 mg/mL; Orion Corporation, Oulu, Finland) was used as a local anesthetic. Anesthetized mice were placed in a Kopf stereotaxic frame equipped with a DKI 921 mouse adapter (David Kopf Instr., Tujunga, CA, USA). The guide cannula was implanted into the right HC using following coordinates: A: −3.1 mm, L: −2.7 mm, V: −1.3 mm and secured with cranial screws and dental cement. Carprofen (5 mg/kg s.c.; Vericore Ltd., Dundee, UK) and buprenorphine (0.12 mg/kg, s.c.; Schering-Plough, Belgium) were used for postoperative pain relief. Saline (0.6 mL i.p.) was injected after surgery to maintain fluid balance. Three hours before the start of the microdialysis experiment, the mice were transferred into microdialysis bowls (CMA120, CMA microdialysis, Solna, Sweden), and the microdialysis probe (membrane length 3 mm, regenerated cellulose; cut-off 18 kDa; Brainlink B.V., the Netherlands) was inserted into the right HC through the guide cannula. The probe was connected to a rotating liquid swivel (375/D/ Characterization of Drug Transporters in AD Models 22QM, Instech Laboratories, Plymouth Meeting, PA, USA) and was perfused with Ringer fluid (147 mM NaCl, 3 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2) at a flow rate of 1.5 μl/ min. After a 120-min wash-out period, a 20 min baseline sample was collected and methotrexate (50 mg/kg in a volume of 2.5 ml/kg; Metoject, medac GmbH) was given intravenously. Dialysate was collected for 240 min (12 samples), and the samples were frozen in liquid nitrogen and stored at −70°C. Terminal blood samples were collected by cardiac puncture and plasma was separated by centrifugation (2000 × g for 10 min at 4°C), immediately fresh frozen on liquid nitrogen and stored at −70°C. In vivo recovery was determined to estimate the true methotrexate concentration in the brain extracellular fluid in a separate experiment (wt n = 3 and APdE9 n = 3, 13 months) by using the retrodialysis method to calibrate the microdialysis probe. The basic procedure of in vivo microdialysis was performed as described above. After a 120-min wash-out period, a 20 min baseline sample was collected and then the Ringer solution was switched to a Ringer containing 200 ng/mL methotrexate. The microdialysis sample collection was continued for 120 min (6 samples). The in vivo recovery for methotrexate was calculated from the last three samples with the eq. C = (Cin-Cout)/Cin, where R is in vivo recovery of methotrexate, Cin is concentration of methotrexate in the perfusate and Cout is concentration of methotrexate in the dialysate. In vivo recovery values of 8 ± 3% for APdE9 mice and 18 ± 7% for WT mice were used to correct the dialysate concentrations. 2655 MTX and the internal standard were 200 V and 160 V, respectively. The collision energies were 38 V and 16 V for MTX and the internal standard, respectively. Agilent MassHunter Workstation Acquisition software (Data Acquisition for Triple Quadrupole Mass Spectrometer, version B.03.01) was used for data acquisition, whereas Quantitative Analysis (B.04.00) software was used for the data processing and analysis. The lower limit of quantification (LLOQ) was 0.05 ng/ml for Ringer fluid samples and 5 ng/ ml for plasma samples. The linearity of the calibration curve was evaluated by a quadratic regression analysis and the method was found to be selective, accurate, and precise over the calibration range. The calibration range of 0.05–500 ng/ ml was used for Ringer fluid samples and 5–1000 ng/ml for plasma samples. Within-run accuracy and precision were calculated from the results of the quality control samples at the three concentrations. The accuracies and precisions for quality control concentrations of 20% were considered acceptable. Data Analysis To evaluate differences in gene expression between brain tissues the statistical significance was tested with Kruskal-Wallis one way analysis of variance on Ranks followed by Dunn’s Method for pairwise comparison (SigmaPlot 13.0). The area under the concentration curve (AUC) values were determined using Phoenix™ WinNonlin® (Pharsight, Certara L.P., USA). In the statistical analysis of differences between AUC values, two-tailed t-test (SigmaPlot 13.0) was used. LC-MS/MS Analysis The microdialysis samples were analyzed for MTX concentration with an Agilent 1200 series liquid chromatograph and an Agilent 6410 Triple Quadrupole Mass Spectrometer with electrospray ionization. The column was a Poroshell 120 ECC18 (50 mm × 2.1 mm, 2.7 μm; Agilent Technologies, Santa Clara, CA) maintained at 40°C and a Rapid Resolution LC in-line filter (2 mm, 0.2 mm; Agilent Technologies) was used for protecting the analytical column from possible contaminants. A binary mobile phase with a gradient elution was used. Solvent A was milliQ water with 0.1% formic acid (eluent additive for LC-MS, Fluka) and solvent B was acetonitrile (Sigma-Aldrich) with 0.1% formic acid. The gradient was performed as following: 5% B kept constant for 1 min, increased to 90% for 5 min, decreased to 5% B for 0.1 min, and kept constant for 2.9 min. Under these conditions, the total run time was 8 min. The flow rate was 0.3 ml/min and the injection volume of Ringer fluid samples was 20 μl. The source parameters were: capillary voltage 3 kV, nebuliser 40 psi, gas temperature 300°C, gas flow 8 l/min. Data acquisition was done in the positive ion MRM mode using transitions 455.1 → 308 for MTX and 458.1 → 311 for the internal standard (deuterated MTX). Fragmentor voltages used for RESULTS Gene Expression Profile of Drug Transporters and Tight Junction-Associated Proteins in Brain of AD Mouse Models Efflux Transporters The expressions of 8 ABC efflux transporters (Abcb1a, Abcb1b, Abcc1-Abcc5, Abcg2) were compared between AD mouse models APdE9, Tg2576 and APP/PS1 (12–16 months old mice) and their healthy age-matched wt controls using brain samples comprising of brain parenchyma and BBB. In the APdE9 model, the greatest difference was detected for Abcb1b gene, which had a 1.4-fold higher expression level in AD CX and HC tissues when compared to wt (Fig. 1a). In Tg2576 mice, the differences between AD tissues and controls were ≤1.4-fold (Fig. 1b) and in the APP/PS1 model, the differences were even smaller (≤ 1.3-fold) (Fig. 1c), if Abcc2 was not taken into account. With respect to Abcc2, up to 2fold differences were detected, but statistical significance was not reached because of the high variation in Abcc2 expression 2656 Kati-Sisko et al. Fig. 1 Gene expression of Abc efflux transporters in the CX, HC and CB of male APdE9 (a), female Tg2576 (b) and female APP/PS1 (c) mouse models expressed as relative to wt control mice (AD/wt) (mean ± sd, n = 3–5). The gene expression was normalized against the beta-actin house-keeping gene. The dotted line depicts 1.5-fold down- or up-regulation. due to its very low expression levels. Overall, the gene expression profile of efflux transporters between these three AD models was similar. Differences between AD and wt were minor (≤ 1.4 fold) with the exception of Abcc2. Influx Transporters Gene expression of SLC influx transporters including L-type amino acid transporter (Slc7a5), peptide transporter (Slc15a2), organic anion and cation transporters (Slc22a2, Slc22a3, Slc22a5 and Slc22a8), Multidrug and toxin extrusion protein (Slc47a1) and organic anion transporting polypeptides (Slco1a4, Slco2b1 and Slco1c1) was studied in the three AD mouse models (Fig. 2). More than 2-fold lower expression in AD samples compared to wt controls was observed for four transporters, namely Slc22a2 (HC of APdE9 and Tg2576), Slc22a3 (CB of Tg2576), Slc22a8 (HC of Tg2576) and Slc47a1 (HC of APdE9 and Tg2576). The highest difference was seen for Slc22a2, the expression of which in HC of Tg2576 mice was approximately one fifth of that in control and a similar trend was also seen in APdE9 (less than half than in controls). The Slc22a8 expression level in the HC of Tg2576 mice was one third of the control and similarly in APdE9, almost half of the control. In APP/PS1 brains, the differences between AD and control were in all cases <1.5 fold. Slc22a2 was expressed at a low level in all studied brain samples; likewise, the levels of Slc22a3 and Slc47a1 were low in certain tissues, thereby introducing high variability into the results for those genes. Tight Junction-Associated Proteins and Cell Markers Four genes (Ocln, Cldn3, Cldn5, Jam1) related to tight junctions were included in the study. Cldn3 expression in HC of Tg2576 and APdE9 mice was lower (0.43 ± 0.14 and 0.61 ± 0.30, respectively) compared to wt control and similarly Jam1 in HC of Tg2576 (0.61 ± 0.11), but in other brain regions and animal models, the differences were smaller (Fig. 3). For the other tight junction-associated genes (Ocln, Cldn5), only minor differences were seen between AD and wt controls. However, with regard to the cell markers analyzed (Pecam1, Aif1, Rbfox3, Gfap), a clear upregulation of the astrocyte marker Gfap was detected. In the CX of APdE9, Tg2576 and APP/PS1 mice, the expression of Gfap was 3.5 ± 0.2-fold (p = 0.037), 2.27 ± 1.13-fold and 4.4 ± 0.2-fold (p = 0.029) higher, respectively, than in wt controls. In addition, in HC, the AD/wt ratio was elevated, i.e. it was 2.4 ± 0.4 in APP/ PS1 and 2.3 ± 0.2 in APdE9. The microglial marker Aif1 expression was upregulated only in APdE9 mice, with 1.6-fold and 1.5-fold higher expressions in HC and CX, respectively. When the expression levels of 26 genes were compared between brain regions (CX, HC and CB) of wt control mice, the differences were more prominent (Table I) and more than Fig. 2 Gene expression of Slc influx transporters in the CX, HC and CB of male APdE9 (a), female Tg2576 (b) and female APP/PS1 (c) mouse models expressed as relative to wt control mice (AD/wt) (mean ± sd, n = 3–5). The gene expression was normalized against the beta-actin house-keeping gene. The dotted line depicts 1.5-fold down- or up-regulation. Characterization of Drug Transporters in AD Models 2657 Fig. 3 Gene expression of tight junctions-associated proteins and cell markers in CX, HC and CB of male APdE9 (a), female Tg2576 (b) and female APP/PS1 (c) AD models expressed as relative to wt control mice (AD/wt) (mean ± sd, n = 3–5). The gene expression was normalized against the beta-actin house-keeping gene. The dotted line depicts 1.5-fold down- or up-regulation. *indicates significant difference between AD and wt (p < 0.05). 2-fold differences were observed for 20 genes. Only Slco1c1 was equally expressed in all three brain regions. Ocln expression was 5–7 -fold higher in CB than in CX, whereas in CX, the expression of Slc22a3 was ≥5-fold higher than in CB and HC. Interestingly, the expression of Abcc1, Slc7a5, Slc22a5, Slc47a1, Cldn5 and Jam1 was higher (>2-fold) in CB than in CX. The differences between brain regions were rather similar among all three wt control strains. Since only minor differences were detected between AD and wt mice, these results are mostly comparable also in AD mice models. Gene and Protein Expression Profile in Brain Microvessel Enriched Fraction of APdE9 Mice The expression of 35 genes was studied in enriched fraction of microvessels isolated from the cortices of 5–12 weeks old APdE9 mice and age-matched controls. In the case of 11 genes, there was a more than 2-fold difference in the expression level between AD and wt (Table II). Only one gene, Slc2a1, was upregulated by 2-fold in AD; for 10 genes, the AD/wt ratio was <0.5. For the endothelial markers Pecam1 and Cdh5, the ratio was in the range 0.31–0.36. Efflux transporters Abcb1a, Abcc4 and Abcg2 Table I Comparison of gene expression between brain regions in three wt strains (healthy control of each AD mice model i.e. male APdE9, female Tg2576 and female APP/PS1) HC/CX APdE9 HC/CX Tg2576 HC/CX APP/PS1 CB/CX APdE9 CB/CX Tg2576 CB/CX APP/PS1 CB/HC APdE9 CB/HC Tg2576 CB/HC APP/PS1 0.88 ± 0.22 0.86 ± 0.20 0.73 ± 0.15 1.72 ± 0.14 1.67 ± 0.29 1.74 ± 0.10 1.94 ± 0.16 1.95 ± 0.34 2.37 ± 0.13 Abcb1a 1.05 ± 0.20 0.91 ± 0.18 1.13 ± 0.19 0.78 ± 0.11 0.48 ± 0.12* 0.69 ± 0.07 0.75 ± 0.11 0.53 ± 0.13 0.61 ± 0.06 Abcb1b 0.95 ± 0.05 0.81 ± 0.18 0.81 ± 0.01 2.37 ± 0.46 2.16 ± 0.22 2.3 ± 0.18 2.5 ± 0.48 2.65 ± 0.27 2.86 ± 0.22 Abcc1 1.12 ± 0.66 0.36 ± 0.16 0.50 ± 0.17 1.15 ± 0.84 0.52 ± 0.59 0.63 ± 0.58 1.03 ± 0.75 1.46 ± 1.65 1.26 ± 1.16 Abcc2 0.90 ± 0.09 0.63 ± 0.13 0.76 ± 0.04 0.61 ± 0.06 0.47 ± 0.27 0.65 ± 0.05 0.68 ± 0.07 0.74 ± 0.43 0.85 ± 0.06 Abcc3 1.13 ± 0.13 0.97 ± 0.21 0.94 ± 0.09 1.97 ± 0.05 1.69 ± 0.31 1.93 ± 0.27 1.75 ± 0.04 1.75 ± 0.32 2.06 ± 0.29 Abcc4 0.68 ± 0.06 0.72 ± 0.05 0.74 ± 0.05 1.37 ± 0.15 1.60 ± 0.21 1.46 ± 0.05 2.01 ± 0.23 2.22 ± 0.30 1.97 ± 0.06 Abcc5 0.87 ± 0.15 0.81 ± 0.2 0.89 ± 0.09 1.31 ± 0.12 1.09 ± 0.17 1.35 ± 0.27 1.52 ± 0.14 1.35 ± 0.21 1.51 ± 0.30 Abcg2 0.85 ± 0.15 0.79 ± 0.17 0.78 ± 0.09 2.21 ± 0.17 2.01 ± 0.41 2.05 ± 0.16 2.58 ± 0.20 2.55 ± 0.52 2.64 ± 0.21 Slc7a5 0.93 ± 0.08 0.96 ± 0.31 1.09 ± 0.18 1.98 ± 0.10 1.66 ± 0.83 2.14 ± 0.11 1.74 ± 0.86 5.54 ± 0.82 5.09 ± 0.75 Slc15a2 1.60 ± 1.09 1.80 ± 1.26 0.97 ± 0.41 1.01 ± 0.26 0.71 ± 0.30 0.88 ± 0.16 0.63 ± 0.16 0.40 ± 0.17 0.90 ± 0.17 Slc22a2 1.21 ± 1.06 0.61 ± 0.07 0.52 ± 0.40 0.15 ± 0.09 0.19 ± 0.05 0.12 ± 0.05 0.18 ± 0.16 0.11 ± 0.01 0.06 ± 0.05* Slc22a3 0.91 ± 0.11 1.14 ± 0.23 1.00 ± 0.11 2.09 ± 0.16 2.46 ± 0.50 2.27 ± 0.20 2.29 ± 0.18 2.16 ± 0.44 2.28 ± 0.20 Slc22a5 1.54 ± 0.78 1.27 ± 0.69 0.92 ± 0.46 1.92 ± 0.30 1.77 ± 0.20 1.20 ± 0.13 1.24 ± 0.19 1.40 ± 0.15 1.30 ± 0.14 Slc22a8 0.90 ± 0.77 1.29 ± 0.79 0.85 ± 0.64 2.70 ± 0.84 2.34 ± 1.28 2.10 ± 0.30 3.00 ± 0.94 1.82 ± 1.00 2.48 ± 0.36 Slc47a1 1.12 ± 0.17 0.95 ± 0.43 0.92 ± 0.12 2.09 ± 0.10 1.48 ± 0.36 1.95 ± 0.13 1.86 ± 0.09 1.55 ± 0.38 2.12 ± 0.14 Slco1a4 0.93 ± 0.08 0.82 ± 0.19 0.93 ± 0.18 0.89 ± 0.13 0.45 ± 0.19 1.00 ± 0.05 0.96 ± 0.14 0.55 ± 0.23 1.07 ± 0.05 Slco2b1 0.89 ± 0.16 0.78 ± 0.11 0.83 ± 0.09 1.17 ± 0.21 1.14 ± 0.12 1.08 ± 0.19 1.33 ± 0.23 1.45 ± 0.29 1.30 ± 0.23 Slco1c1 1.76 ± 0.15 2.55 ± 0.62 1.74 ± 0.15 3.20 ± 0.09 2.84 ± 0.30 3.09 ± 0.23 5.64 ± 0.16 7.25 ± 0.75* 5.37 ± 0.40 Ocln 1.30 ± 1.04 1.87 ± 1.94 1.09 ± 0.16 1.49 ± 0.80 0.89 ± 0.50 1.39 ± 0.94 1.14 ± 0.62 0.47 ± 0.27 1.28 ± 0.87 Cldn3 1.24 ± 0.25 1.14 ± 0.30 0.88 ± 0.33 1.88 ± 0.17 2.12 ± 0.84 1.43 ± 0.28 1.51 ± 0.13 1.86 ± 0.74 1.62 ± 0.32 Cldn5 1.05 ± 0.18 1.30 ± 0.96 0.99 ± 0.27 2.38 ± 0.25* 3.78 ± 1.54 1.65 ± 0.29 2.27 ± 0.24 2.91 ± 1.19 1.67 ± 0.30 Jam1 1.11 ± 0.29 1.06 ± 0.35 0.99 ± 0.27 1.86 ± 0.09 1.44 ± 0.44 1.73 ± 0.17 1.67 ± 0.08 1.36 ± 0.42 1.74 ± 0.17 Pecam1 0.76 ± 0.08 0.72 ± 0.14 0.84 ± 0.11 0.80 ± 0.14 0.53 ± 0.18 0.51 ± 0.02 1.06 ± 0.18 0.74 ± 0.25 0.60 ± 0.02 Aif1 0.63 ± 0.08 0.73 ± 0.02 0.75 ± 0.02 1.59 ± 0.30 2.18 ± 0.16 2.09 ± 0.23 2.54 ± 0.48 2.97 ± 0.22 2.80 ± 0.30 Rbfox3 2.18 ± 0.29 2.23 ± 0.50* 2.38 ± 0.25 1.68 ± 0.16 1.85 ± 0.22 2.67 ± 0.45 0.77 ± 0.07 0.83 ± 0.10 1.12 ± 0.19 Gfap Results are expressed HC/CX, CB/CX OR CB/HC ratio (mean ± sd, n = 3–5). Yellow color indicates ≥1.5-fold and green color ≥2-fold differences between brain regions. * = p < 0.05 2658 Kati-Sisko et al. had 73%, 60% and 55% lower expression levels, respectively, in AD than in wt. A similar trend was observed in case of the expression of the influx transporters Slc22a8 (55%) and Slco1a4 (61%), beta-amyloid peptide receptor Ager (Rage) (65%) and tight junction-associated genes occludin (60%) and claudin 5 (64%). Results were normalized against beta-actin, which showed stable expression between AD and wt samples. Since the amount of RNA was limited, the replicate measurements were focused on genes with moderate or high expression and/or where a difference between AD and wt was detected in the first measurement. Table II Distribution of Methotrexate into Brain in APdE9 Mice To assess the effect of AD pathology on efflux transporter function in vivo, methotrexate was selected as a model drug for the Comparison of Gene Expression Levels between Brain Microvessels Isolated from Cortices of Female AD Mice (APdE9) and their wt Controls ATP-binding cassette (ABC) transporters Gene Abcb1a Abcb1b Abcc1 Abcc2 Abcc3 Abcc4 Abcc5 Abcg2 Solute carriers (SLCs) Slc2a1 Slc5a7 Slc7a5 Slc15a2 Slc16a1 Slc22a2 Slc22a3 Slc22a5 Slc22a8 Slc29a1 Slc47a1 Slco1a4 Slco2b1 Slco1c1 Transport of beta-amyloid Lrp1 Ager Tight junction-associated proteins Ocln Cldn1 Cldn3 Cldn5 Tjp1 F11r Cell markers Pecam1 Cdh5 Aif1 Gfap Rbfox3 The microvessel enriched fraction was used also to study the protein expression of the main receptors for amyloid-beta peptide, Rage and Lrp1, with the efflux transporters Mdr1 and Mrp4 showing lower gene expression in AD than in wt mice. According to the results of immunoblotting, the expression of these proteins did not differ significantly (≤ 1.5-fold) between AD and wt samples (Fig. 4). Protein Multidrug resistance protein 1a (Mdr1a) Multidrug resistance protein 1b (Mdr1b) Multidrug resistance-associated protein 1 (Mrp1) Multidrug resistance-associated protein 2 (Mrp2) Multidrug resistance-associated protein 3 (Mrp3) Multidrug resistance-associated protein 4 (Mrp4) Multidrug resistance-associated protein 5 (Mrp5) Breast cancer resistance protein (Bcrp) Ratio of AD/wta 0.258–0.283 0.537 0.758 0.677 1.011 0.3971–0.3973 0.588–0.668 0.436–0.457 Glucose transporter 1 (Glut1) High affinity choline transporter 1 (Cht1) L-type amino acid transporter 1 (Lat1) Peptide transporter 2 (Pept2) Monocarboxylate transporter 1 (Mct1) Organic cation transporter 2 (Oct2) Organic cation transporter 3 (Oct3) Organic cation/carnitine transporter 2 (Octn2) Organic anion transporter 3 (Oat3) Equilibrative transporter 1 (Ent1) Multidrug and toxin extrusion protein 1 (Mate1) Organic anion transporting polypeptide 1a4 (Oatp1a4) Organic anion transporting polypeptide 2b1 (Oatp2b1) Organic anion transporting polypeptide 1c1 (Oatp1c1) 1.803–2.230 1.173–1.653 0.696–0.943 0.766–0.913 0.620–0.657 1.161 0.990 0.626 0.414–0.495 0.941–1.03 0.543 0.369–0.405 0.714–0.726 0.525–0.563 Full name Low density lipoprotein receptor-related protein 1 Advanced glycation end product-specific receptor (Rage) 0.960–0.964 0.300–0.400 Occludin Claudin1 Claudin 3 Claudin 5 Tight junction protein ZO-1 Junctional adhesion molecule A (Jam-A) 0.362–0.431 1.079–1.334 1.052 0.308–0.410 0.555–0.646 0.591–0.666 Full name/Cell type Platelet/endothelial cell adhesion molecule 1/Endothelial Cadherin-5/Endothelial Allograft inflammatory factor 1/Microglial Glial fibrillary acidic protein/Astrocytic RNA binding protein, fox-1 homolog (C. elegans) 3/Neuronal 0.324–0.363 0.306–0.354 0.826–0.898 1.07–1.09 1.243 a When results are expressed as range, two replicate samples were used. Otherwise the results are based on one sample of isolated microvessels. Each sample is a pool from brain microvessels of 5 mice Characterization of Drug Transporters in AD Models brain microdialysis studies since it has been previously shown to be effluxed by both Mrp4 and Bcrp in a similar study conducted in Mrp4 and Bcrp knock-out mice (23). Unbound extracellular methotrexate levels in hippocampus were 9-fold higher in APdE9 mouse than the corresponding values in wt in the 20 min fraction after drug administration (Fig. 5), but the difference diminished later. The brain methotrexate exposure (AUC 0-∞ ) was 2.7-fold higher in APdE9 than in wt (p = 0.013) mice and the difference was even greater, 2.9-fold (p = 0.006), within the first 60 min (AUC0–60) (Table III). Total plasma concentration was measured after intravenous injection of methotrexate from separate mice (wt n = 6 and APdE9 n = 5–6, 17 months). The plasma concentrations at 10, 30 and 60 min were 52.7 ± 7, 14.0 ± 7.2 and 5.9 ± 1.1 μg/ml (mean ± sd) in APdE9 and 58.4 ± 16, 14.1 ± 4.5 and 6.6 ± 2.6 μg/ml in wt mice, respectively. DISCUSSION Transporters and tight junctions of the BBB are crucial for the maintenance of brain homeostasis. Neurodegenerative diseases, such as AD, alter the function of the BBB and are therefore likely to change drug distribution into the brain (24). In this study, the expression of Abc and Slc transporters relevant in pharmacokinetics and tight junction-associated proteins in the brains of three AD mouse models, APdE9, Tg2576 and APP/ PS1 were investigated. We focused on genes that are known to be expressed in brain/BBB. Brain samples were dissected from >12 months old animals having clear pathological signs of the disease in their brain i.e. amyloid beta plaques and gliosis. We used APP and APP/PS1 mutant mouse lines considered to be AD models of Aβ pathology rather than AD models fully addressing the clinical presentation of AD. The expression levels of the studied genes in AD models were close to their healthy age-matched controls and the observed differences were minor 2659 Fig. 5 Unbound extracellular methotrexate levels in the hippocampus of male APdE9 mice (AD) (n = 7) and wt controls (n = 8) after i.v. injection at 50 mg/kg. Microdialysis samples were collected as 20 min fractions. LLOQ was 0.05 ng/ml (mostly less than 1.5-fold change) with the exception of the astrocyte cell marker, Gfap. This was expected since upregulation of Gfap due to an increase in the numbers of reactive astrocytes is an early and common sign in various models of neurodegeneration and brain diseases, and is also observed in transgenic AD mice (25). Furthermore, the expression of the microglial activation marker, Aif1, was elevated (about 1.5fold) in APdE9 mice in line with extensive data revealing neuroinflammation as a characteristic of AD. Comparison of gene expression of transporters and tight junction-associated proteins between brain regions showed more than 2-fold differences for 21 genes in wt mice. For 14 genes, a higher expression level was observed in CB in comparison to CX or HC. We detected a > 5-fold difference between brain regions in gene expression of organic cation transporter Slc22a3 and the tight junction-associated protein, occludin. Such large differences in gene expression should reflect protein expression and functionality. In the literature, the data regarding regional variability of brain drug transporters is sparse and scattered. The highest Mdr1 activity has been proposed for CB by a combinatory mapping approach and PET studies in rats (26,27). These observations are in accordance with our data as Abcb1a is the major form in brain. However, in another study, Mdr1 mediated efflux activity of loperamide was highest in cerebral CX and midbrain regions when assessed by in situ brain perfusion in mice (28). Kannan et al. stated that there was equal BCRP density Table III Brain Exposure to Methotrexate in Male APdE9 Mice (AD) and their wt Controls After i.v. Injection at 50 mg/kg. AUC Calculated from Whole Concentration-Time Curve (AUC0-∞) and First 60 min (AUC0–60) are Presented (mean ± SEM, n = 7–8) Fig. 4 Expression of Rage, Lrp1, Mdr1 and Mrp4 proteins in microvessel enriched fraction isolated from cortices of female APdE9 (AD) and wt mouse and detected by Western blotting. For Rage, Lrp1 and Abcb1 40 μg of total protein were used, the corresponding value for Mrp4 was 5 μg. Mean fold change ± SEM (AD/wt) was calculated based on three replicate blots AUC0-∞ (ng x min/ml) AUC0–60 (ng x min/ml) *p < 0.05 vs. wt control value **p < 0.01 vs. wt control value AD wt control 40178 ± 7587* 27485 ± 4167** 15107 ± 4765 9507 ± 3604 2660 between brain regions, which is also in line with our results (13). The high regional variability in transporter expression may perhaps be explained by the higher capillary density in certain brain region or the variation in the relative amount of each cell type. Previously, brain capillary density has been determined with the vascular volume marker, inulin, with a 3.7-fold difference being observed between brain regions (28). This high variation between brain areas can affect the regional distribution of drugs in brain, but more information about transporter expression at the cell type level will be needed to estimate the effect of regional variability on brain pharmacokinetics and access of drugs to their target site. The endothelial cells of the BBB and tight junctions expressed between these cells form the physical barrier for brain distribution of drugs. Since the brain microvessels comprise only 0.1% of brain weight and brain tissue contains many cell types (endothelial cells, astrocytes, microglial cells, neurons and pericytes), the alterations in gene and protein expression occurring solely in the microvessel endothelial cells can be interfered by other cell types. Therefore, further studies to reveal AD-induced changes in BBB protein expression were conducted by using brain microvessels isolated from APdE9 mice. The isolation of a microvessel enriched fraction is feasible only in young mice (≤12 weeks), whose brain pathology of AD is still developing. The normalized expressions of endothelial markers, Pecam1 and Cdh5, were about 3-fold lower in APdeE9 compared to their healthy control mice, suggesting that APP and PSEN1 mutations may considerably affect the brain vasculature already during development or in young adulthood. Both increased and decreased vascular densities have been reported in AD (29). The gene expression of Abcb1a was downregulated in the microvessel enriched fraction which is in line with the earlier reports from AD patients and animal models (8,10). Furthermore, Abcc4 and Abcg2 were downregulated. In order to assess the effect of AD pathology on efflux transporter function, MTX was selected as a model drug, because it has been previously demonstrated to cross the BBB via Mrp4 and Bcrp in a brain microdialysis study conducted in Mrp4 and Bcrp knock-out mice (23). Methotrexate is also a substrate for MDR1, OAT and OATP transporters (30–34). Since Abcb1a (Mdr1a), Slac22a8 (Oat3) and Slco1a4 (Oatp1a4) were also downregulated in the microvessel enriched fraction, it is possible that these transporters participate in the distribution of methotrexate into the brain. As far as we are aware, this is the first study where AD-induced changes in brain unbound extracellular drug concentrations have been studied by pharmacokinetic microdialysis in the context of transporter function. Brain microdialysis revealed that methotrexate gained faster access into the brain in 16–18 months old APdE9 mice after drug administration but the difference vs. wt controls diminished later. This finding may reflect alterations in the active transport in BBB or otherwise compromised BBB. Identification of functionally Kati-Sisko et al. altered transporter(s) is challenging due to overlapping substrate specificities and would require experiments with inhibitors. The consequences of BBB disruption, even when local or transient, can be harmful for the homeostasis of brain. Aβ has been reported to compromise BBB tight junction proteins and thus BBB integrity in human amyloid precursor protein transgenic mice (35) and human apoE4 transgenic mice (36). Generally, in tumor-bearing rats, a compromised BBB has been shown to increase brain extracellular MTX levels shortly after drug administration (37). However, Bien-Ly et al. (2015) reported recently that brain access of antibodies and radiotracers was not significantly altered in AD mouse models (38). Cheng et al. (2010) also reported that brain penetration of some small-molecule drugs was unaltered in AD-related animal models and Mehta et al. (2013) even found decreased brain uptake of transcellular permeability markers (diazepam and propranolol) in the 3xTg model possibly due to thickening of the basement membrane (16,39). Furthermore, imaging studies with AD patients have not found convincing evidence that AD alone, without a vascular component, causes BBB disruption (2). These discrepancies both in experimental models of AD and patients may partly be due to variation in the degree of AD pathology and the characteristics of the model drugs. Accumulation of Aβ plaques within the extracellular space, severe shrinkage of cerebral cortex and hippocampus, and an increase in the size of the ventricles in AD brains may affect the distribution of drugs in brain tissue. For a hydrophilic drug like methotrexate (LogDpH7.4–5.22, Chemspider, predicted using ACD/Labs), a smaller volume of extracellular fluid can lead to higher initial drug concentration in AD mice after a single systemic dose. No significant differences were detected between <3 month old APdE9 mice and their controls at the protein level in the expression of Mdr1, Mrp4, Rage and Lrp1 in the microvessel enriched fraction. Earlier, Hartz et al. (2010) demonstrated that Mdr1 expression was decreased by approximately 60% and efflux of Mdr1 substrate NBD-CSA was reduced by 70% in brain capillaries of Tg2576 mice (8). Recently, Do et al. (2016) stated that Abcb1 and Abcg2 expressions in the brain capillary fraction of another AD model, 3xTg-AD were upregulated by 1.7-fold at 18 months of age, although younger mice (3 and 6 months) did not display significant changes (17). With the same AD model, Mehta et al. reported that cortical or hippocampal uptake of three Mdr1 substrates, digoxin, loperamide and verapamil, was not significantly altered in 18–20 month old 3xTg-AD mice (16). However, Mdr1 expression showed variability between replicates, ranging from a 42% reduction to no difference in 3xTg-AD compared to wt controls. These results demonstrate that alterations in efflux proteins, induced by AD-causing mutations, are minor in animal models but comparable to those encountered in human studies. The expression level of transporters affects the maximum velocity of transport (Vmax) and therefore, it can be assumed to reflect in transport function. A decrease in the transporter Characterization of Drug Transporters in AD Models expression is comparable to the inhibition kinetics of active transport, where the degree of inhibition increases with inhibitor concentration (40). Should efflux transporter expression decrease by 50%, this would cause at maximum a 2-fold increase in the steady state drug concentration in the brain, assuming that the drug passes through the BBB exclusively via this specific transporter. Similarly, a 67% decline in expression would cause at maximum a 3-fold and an 80% decline would evoke a maximum of a 5-fold increase in brain drug concentration at steady state. If we consider the clinical relevance of alterations detected in AD patients, mostly 2-fold or smaller alterations for MDR1 have been observed. For instance, when the activity of MDR1 was studied with PET imaging, the binding potential of verapamil was found to be modestly increased in AD patients (2.18 ± 0.25) compared to healthy controls (1.77 ± 0.41) (14). In addition, the expression of MDR1 was 25% lower in the blood vessels of AD patients than age–matched controls when hippocampal sections were studied by immunofluorescence (10). Accordingly, when Kannan et al. recently studied transporter density in the vascular space, they found that MDR1 global density was 53% lower in the temporal CX of AD patients as compared to controls, and in capillaries near to Aβ deposits, the MDR1 density was locally decreased by 35% (13). Our present results with AD models are in line with these observations. Therefore, in order that a detected approximately 2-fold change in expression level should be clinically significant, the drug should have an extremely narrow therapeutic level and adverse effects in brain should be the dose-limiting factor, an extremely rare situation for clinically used drugs (40). It should be kept in mind that in addition to the expression level, transporter functionality is also dependent on other factors such as post-translational modifications and changes in membrane composition, which may change in Alzheimer’s disease and affect binding affinity and transport activity. Furthermore, binding to plasma proteins is an important factor influencing drug distribution into the brain since only unbound drug is able to cross the endothelial cell barrier. Disease states can change the plasma protein binding, but thus far, no significant changes in plasma protein content have been observed in AD patients. In addition to the influence of disease, other factors such as age and sex may affect transporter expression (41, 42). However, in this study, we used age-matched controls of the same gender in order to assess the effect of AD on drug transporters. In conclusion, transporter expression varied significantly between brain areas in both transgenic AD mice and wt mice. However, disease-induced alterations in transporter expression were low in all studied transgenic AD mouse models, which accords with the current knowledge on diseaseinduced changes in AD patients. Decreased expression of some transporters and tight junction-associated proteins in brain microvessel fraction of APdE9 mice as well as more 2661 rapid brain access of MTX in APdE9 mice indicate that AD pathology may contribute to the BBB functionality to some extent. ACKNOWLEDGMENTS AND DISCLOSURES This study was funded by Academy of Finland (Grant number 257386). Finnish Cultural Foundation is also acknowledged for financial support. KMK and TM acknowledges the funding from the Academy of Finland and Sigrid Juselius Foundation. We thank Dr. Aaro Jalkanen for advice in designing microdialysis experiment, Prof. Seppo Auriola for consultation in LC-MS/MS analysis and Lecturer VeliPekka Ranta for advice regarding pharmacokinetics. Mrs. Jaana Leskinen, Mrs. Leena Pietilä and Mrs. Lea Pirskanen are acknowledged for technical assistance. The authors declare that they have no conflict of interest. All applicable international, national and institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the University of Eastern Finland or practice at which the studies were conducted. This article does not contain any studies with human participants performed by any of the authors. REFERENCES 1. 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ISSN impresa ISSN online Maderas. Ciencia y tecnología 2023 (25): 28, 1-16 0717-3644 0718-221X DOI: 10.4067/s0718-221x2023000100428 WOOD QUALITY OF TEN CLONAL PROGENIES OF RUBBER TREE AS RAW MATERIAL FOR BIOENERGY Erick Phelipe Amorim1,♠ https://orcid.org/0000-0002-8964-0770 Eduardo Luiz Longui2 https://orcid.org/0000-0002-7610-9147 Miguel Luiz Menezes Freitas2 https://orcid.org/0000-0001-8882-0908 Fábio Minoru Yamaji1 https://orcid.org/0000-0002-0908-8163 Francides Gomes da Silva Júnior3 https://orcid.org/0000-0002-9142-7442 Marcela Aparecida de Moraes4 https://orcid.org/0000-0003-3572-9291 José Cambuim4 https://orcid.org/0000-0003-0839-633X Mario Luiz Teixeira de Moraes4 https://orcid.org/0000-0002-1076-9812 Paulo de Souza Gonçalves5 https://orcid.org/0000-0002-5019-1832 ABSTRACT The use of wood as a bioenergetic source requires knowledge of its technical properties. The rubber tree Hevea brasiliensis has an economic life cycle of 25 to 30 years in Brazil. It is used for extracting rubber and generating residual wood for fuel. Our goal was to characterize the wood quality of 10 clonal progenies as a source of raw material for bioenergy. Ten clonal progenies of 12-year-old Hevea brasiliensis from an experimental planting in Selvíria municipality were evaluated. Three trees per clone were evaluated for individual properties of Higher Heating Value, immediate chemical analysis, chemical composition, fiber diUniversity Federal of São Carlos. São Carlos, Brazil Forestry Institute of São Paulo. São Paulo, Brazil 3 School of Agriculture Luiz of Queiroz. São Paulo, Brazil 4 University State Paulista. São Paulo, Brazil 5 Institut Agronomic of Campinas. São Paulo, Brazil ♠ Corresponding author: erick.amorim95@hotmail.com Received: 05.12.2020 Accepted: 28.03.2023 1 2 1 Maderas. Ciencia y tecnología 2023 (25): 28, 1-16 Universidad del Bío-Bío mensions, thermogravimetric analysis of wood under nitrogen atmosphere and Fourier Transform Infrared Spectroscopy analysis. We highlight clone IAC 311 for fuel because it presents elevated Higher Heating Value and fixed carbon and less volatile material. However, the other genetic materials studied also meet the specifications for energy use and can be highly viable given their physical, chemical, energy, and thermal properties. Keywords: Raw material, bioenergy, chemical composition, Hevea brasiliensis, Fourier Transform Infrared Spectroscopy, thermogravimetric biomass, wood density. INTRODUCTION The reduction of fossil fuels, especially oil, natural gas and coal, was discussed by Gonçalves et al. (2015), while their substitution by renewable energy sources was suggested by Mehmood et al. (2017). The generation of energy through wood can be a good alternative to replace the energy generated by fossil fuels, since it emits less greenhouse gases in the combustion process, considering that the use of fossil fuels leads to numerous problems to the environment, which is caused by excess CO2 in the atmosphere, including: acid rain, caused by the reaction between pollutants and water vapor in the atmosphere; gradual decrease of the ozone layer and water contamination (Skeva et al. 2014). Vegetal biomass can be used to obtain the most varied forms of energy, whether by direct or indirect conversion. The advantages of using this material as a substitute for fossil fuels include less atmospheric pollution and the stability of the carbon cycle. Compared to other types of renewable energy, vegetable biomass stands out for its high energy density and the ease of storage, conversion, and transport of this material (Saccol et al. 2020). The internal supply of energy in Brazil consists of 43,5 % of renewable sources, of which biomass corresponds to 9,8 % Empresa de Pesquisa Energética-EPE (2022). According to data from Brazilian tree industry (2023), areas with forests planted for industrial purposes in Brazil totaled 9,9 million in 2022 and contributed 91 % of all wood produced for industrial purposes. Species and clones of Eucalyptus and Corymbia are widely used as sources of biomass in Brazil (Tenorio and Moya 2013). However, knowledge of other forest species and clonal progenies, such as Hevea brasiliensis, can be an alternative to increase the use of forest biomass and reduce dependence on fossil fuels in the energy matrix, thus contributing to the country’s economic development. Hevea brasiliensis is an important forest species because its main output is latex, which is responsible for the fabrication of several synthetic products for industrial use. However, when rubber trees no longer reach acceptable levels of latex production, which varies between 25 and 30 years of age (Lima et al. 2020), their exploitation becomes economically unfeasible. Consequently, these trees are felled for reformulation of planting, and, in general, the wood does not have an adequate destination, other than use as firewood in small rural locations. No thought has been given to its energetic characteristics for industrial use (Ramos et al. 2016). Since 1995, the Brazilian State of São Paulo has established itself as the main rubber producer, representing half of the country’s production (Brioschi et al. 2010). The planted area in Brazil has been increasing significantly, from 159,500 ha to 218,307 ha in 2022 alone (IBÁ 2023). This increase in the cultivable areas of the species in Brazil, was due to the potential of the rubber tree species in capturing CO2 and other gases harmful to the environment and the use of the species in agroforestry systems (Satakhun et al. 2019). Thus, the species has potential for latex extraction, helps to balance the environment, and is a source of renewable biomass for clean energy production. According to Ratnasingam et al. (2009), aside from latex, H. brasiliensis produces a large amount of biomass, an estimated 2,1 m³ from each tree at 25 years old with a tree diameter of 40 cm and 20 m in height. Therefore, it is well past time to consider identifying alternative uses of this biomass, as well as study of this waste to produce energy, which would help reduce dependence on fossil fuels with implications for the environment Bersch et al. (2017). Wood quality evaluation for energy purposes aims to measure and classify the superior characteristics of clonal progenies because such evaluation is essential to decision-making in the implementation of reforestation projects aimed at the use of biomass as an energy source. Wood characteristics provide a way to differentiate among different clone types (Lima et al. 2011). According to Protásio et al. (2019), the main wood properties for the selection of energy species are basic density, higher heating value, lignin content, ash contents, fiber 2 Maderas. Ciencia y tecnología 2023 (25): 28, 1-16 Wood quality of ten clonal progenies of rubber tree..: Amorim et al. wood anatomy and thermal behavior. Among wood properties, basic density is considered one of the most important parameters among physical wood properties, as it affects most other properties. Its effects, however, are interactive and difficult to assess in isolation, requiring knowledge of other quality wood properties for the selection of superior clonal genotypes (Pereira et al. 2016). According to Purba et al. (2021), forest biomass quality for energy production varies among genetic materials between and within wood, thus it is important to evaluate wood quality to validate its suitability for use. Scriba et al. (2021), cite that the study of wood properties among clones with potential employment in the forestry market is important for the selection and recombination of superior genetic material from the best individuals with the best quality of the desired technological properties for the establishment of better-quality plantations for the desired use. Chemical and anatomical composition of wood is also relevant for energy use. Specifically, when wood is thermally degraded, it undergoes a transformation process whereby all its primary components, including cellulose, hemicellulose, and lignin, are drastically altered, in turn affecting its energetic properties (Yu et al. 2017). The immediate chemical analysis of an energy source provides a profile of volatile material contents, fixed carbon, and ash (residual material), all of which influence the properties of burning fuel because volatile constituents burn quickly, and fixed carbon burns slowly (Fernandes et al. 2013). The content of volatile materials and fixed carbon is causally related to calorific value, increasing the burning time of the energy source. The content of volatile materials and fixed carbon in wood are interdependent properties since the percentage of ash thereof is generally low (Silva et al. 2020). Thermal stability is an essential consideration when selecting biomass with energy potential. Techniques, such as thermogravimetric analysis (TGA), make it possible to understand biomass decomposition as a function of heating in thermochemical conversion processes. Thermogravimetric analysis consists of analyzing variation of sample mass using a system with controlled temperature and atmosphere. In addition to obtaining information on composition and thermal stability, this analysis provides an assessment of temperature ranges at which decomposition is most pronounced (Yeo et al. 2019). The evaluation of energy properties and efficiency of material allows the selection of more competitive materials compared to other energy sources. In this context, our objective was to characterize the quality of the wood from 10 clones of Hevea brasiliensis, as a source of raw material for bioenergy through the characterization of the anatomical properties of fiber length and width, physical property basic density of the wood: energy properties through the characterization of the higher heating value, immediate analysis through the contents of extractives, lignin and holocellulose. We also evaluated the chemical characterization of the main chemical groups of wood through FTIR- Fourier Transform Infrared Spectroscopy, and biomass characterization through thermal degradation by thermogravimetric (TGA) analysis. MATERIALS AND METHODS Selection of species and planting Wood samples were collected from 30 with 12-year-old rubber Hevea brasiliensis (Willd. ex Juss.) Muell. Arg. trees (Table 1), three of each clonal progeny, in the municipality of Selvíria, Mato Grosso do Sul State (20°20′S, 51°24′W, elevation 350 m). The trial plantation was established in 2006 at a spacing of 3 m × 3 m from seeds of 10 free-pollinated clones (IAC 40ill., IAC 41ill., IAC 326ill., IAC 311ill., IAC 301ill., IAN 873ill., GT1ill, PB 330ill., Fx 2261ill., and RRIM 725ill.). Soil in the experimental area was classified as Red Latosol with clayey texture (Santos et al. 2006). In 2018, three selected trees of clonal progenies were felled, and discs 10 cm in thickness from each tree at breast height (1,30 m from the ground) were cut from each tree. From each disc, samples close to the bark were used to determine HHV, chemical constituents, wood density, fiber dimensions, thermogravimetric parameters, and properties under Fourier-Transform Infrared Spectroscopy- FTIR. 3 Maderas. Ciencia y tecnología 2023 (25): 28, 1-16 Universidad del Bío-Bío Table 1: Improved population consisting of ten open-pollination progenies. ill.= illegitimate (progenies obtained from a free-pollination clone). Determination of chemical and energy properties Wood was ground in a Wiley knife mill, of metallic steel type, manufactured by the Marconimaterials company, with the company located in the municipality of Piracicaba, in the state of São Paulo, Brazil, transformed into sawdust, and then sieved with 40-60 mesh to later characterize its chemical and energetic properties. Higher heating value Samples were fragmented into smaller pieces with a hammer and chisel and milled in a micro mill. The samples, before the determination of the Higher Heating Value (HHV), were dried in greenhouses to determine the moisture content. To perform the analysis, the isoperibolic method was used with an IKA C200, calorimeter of the type coated plastic from the company Biovera-Laboratory Equipment and Technical Assistance, with its company based in Rio de Janeiro, in the state of Rio de Janeiro, Brazil. the methodology adopted for the determination of the HHV, according to ASTM D5865-98 (2004). Proximate analysis Prior to these analyses, the biomasses (all treatments) were dried in an oven at 100 °C for 10 minutes to get the moisture to 0 %. The determination of ash content was performed according to the standard ASTM D1102-84 (2013), and the volatile content was determined according to ASTM E872 (1982). Both tests were done in triplicate. Both standards were adopted, as all material was used for the calculation. The fixed carbon content was calculated according to Equation 1. FCC =100 − ( AC + VC ) (1) Where: FCC= Fixed Carbon Content (%); AC= Ash Content (%); VC= Volatile Content (%). Chemical assays To determine extractives (EX) and lignin (LI) contents, TAPPI T204 (2001) and TAPPI T22 (2011) were used, respectively. The samples were fragmented into smaller pieces with a hammer and chisel and milled in a micro mill. The resulting powder was sieved through 40 mesh and 60 mesh screens, and the material retained on the last sieve was used for analysis. The analyses were sequential such that the extractives were 4 Maderas. Ciencia y tecnología 2023 (25): 28, 1-16 Wood quality of ten clonal progenies of rubber tree..: Amorim et al. first removed, then lignin by acid treatment, with holocellulose (HO) content finally calculated. For extractive contents, extractions were performed in solutions of toluene: alcohol (2:1v:v) and alcohol, at times exceeding 12 h in a Soxhlet extractor. For lignin, extractive-free powder was prepared in several stages with 72 % sulfuric acid to obtain insoluble and soluble lignin (Cary 100 UV-visible spectrophotometer). Finally, the two lignin values were added. The content of insoluble lignin (IL) was determined according to Equation 2.  DWlig  IL =   x 100 (2)  DW  Where: DW=dry sawdust weight, and DWlig=dry weight of insoluble lignin. Soluble lignin (SL) filtrates and the blank were read at two wavelengths (215 nm and 280 nm), respectively, using quartz cuvettes. The soluble lignin (SL) content was determined according to Equation 3. SL = ( 4,53 ( L215 − blank ) − L280 − blank ) x 100 ( 300 DW ) (3) Where: DW=dry sawdust weight. Ex and Li were expressed as a percentage (%) of oven-dried weight of unextracted wood. After determining the levels of extractives, soluble and insoluble lignin, the holocellulose (HO) content was calculated according to Equation 4. HO = (100 − ( Ex + Li ) ) (4) Where: Ex=Extractives and Li=Lignin. To determine each variable, a triplicate was used for each clone of Hevea brasiliensis. Wood density Wood density was determined by the ratio between dry mass and saturated volume, according to Brazilian standard ABNT NBR-11941 (2003). Fiber analysis Small pieces were cut from the side of samples, and macerations were prepared according to the modified Franklin method (Berlyn and Miksche 1976). Modifications resulted from the differences caused by the higher concentration of hydrogen peroxide used in our study. Macerations were stained with alcoholic safranin and mounted in a solution of water and glycerin (1:1). Fiber measurements were performed on an Olympus CX 31 microscope equipped with a camera (Olympus Evolt E330) and a computer with image analyzer software (Image-Pro 6.3). Terminology followed the IAWA list (1989). Fiber length (FL) and fiber wall thickness (FWT) were evaluated. 5 Maderas. Ciencia y tecnología 2023 (25): 28, 1-16 Universidad del Bío-Bío Thermogravimetric analysis (TGA) Sieved samples consisted of remnants retained in the 30-mesh sieve. Approximately 20 mg of clonal material from each progeny was heated from 0 ºC to 700 ºC at 20 ºC/min-1 under nitrogen atmosphere, using TGA 55 equipment. The degradation analysis was performed for each thermogravimetric event of rubber tree clones. From TG curves, mass loss calculations were performed in the following temperature ranges: 50 ºC - 100 °C, 100 ºC - 250 °C, 250 ºC - 400 °C, 400 ºC - 600 °C and 600 ºC - 700 °C. The residual mass at 700 °C was also calculated for each clonal progeny of Hevea brasiliensis. Fourier transform infrared spectroscopy (FTIR) Sieved samples consisted of remnants retained in the 30-mesh sieve. Approximately 10 mg of the material were used to read the absorbance spectra using a Perkin Elmer model Spectrum 65 spectrometer in the region from 500 cm-1 to 4000 cm-1, spectral resolution of 4 cm-1 and 32 scans. Data analysis Tests of homogeneity and analysis of variance (ANOVA) were performed, and when significant difference was detected between treatments, the Tukey test was used, at 1 % significance. The data were analyzed with the statistical software SigmaPlot version 12. RESULTS AND DISCUSSION Chemical and energetic properties, basic density and fiber analysis Table 2 shows the values for the energetic properties for each of 10 rubber clonal progenies. The values of HHV ranged from18357 kJ·kg-1 (IAC-301ill.) to 19070 kJ·kg-1 (IAC-311ill.), fixed carbon from 15,16 % (IAC-40ill.) to 15,72 % (IAC-311ill.), volatile material from 82,79 % (IAC-311ill.) to 83,92 % (IAC-301ill.), and ash content from 0,42 % (IAC-311ill.) to 1,49 % (IAC-301ill.). The values oscillated among the different rubber tree clonal progenies, presenting different values in their energetic and chemical properties. The values of energy and chemical characteristics, wood density and wood fiber of rubber tree clonal progenies showed different behaviors. Progenies IAC 311ill., PB 330ill., IAC 41ill., IAC 301ill., and IAN 873ill. presented higher lignin and fixed carbon levels that contributed to HHV (Table 2), thus presenting superior energy performance compared to other clones. According to Trugilho and Silva (2001), these differences in energy properties can be associated with chemical composition that can affect the energy characteristics of biomass. However, Muzel et al. (2014) reported Brazilian forest species that presented higher HHV and were, therefore, widely used for energy generation, as noted by the author above. Studying the wood of clones of Eucalyptus grandis and H. brasiliensis, Telmo and Lousada (2011) reported HHV of 17895 kJ·kg-1 and 17897 kJ·kg-1. Such similarity between these species highlights the wide use of Eucalyptus in Brazil for the generation of energy. 6 Maderas. Ciencia y tecnología 2023 (25): 28, 1-16 Wood quality of ten clonal progenies of rubber tree..: Amorim et al. Table 2: Comparison among ten 12-year-old rubber tree clonal progenies, according to energetic properties, chemicals, wood density and fiber analysis. ill. = illegitimate (progenies obtained from a free-pollination clone); HHV= Higher heating value; CC = carbon content; VMC = volatile matter content; AC = ash content; EC = extractives content; LC = lignin content; HC = holocellulose content; ρbas = Wood density; FL= fiber length; FWT = fiber wall thickness. Distinct letters in rows differ statistically (P<0,001) by Tukey’s test. For decades, HHV of wood for power generation was considered 18830 kJ·kg-1. HHV differences between clones observed in this study can be explained by the ecological group of the wood (Tan 1989). Among hardwoods, rubber tree clones have expected HHV of 19089 kJ·kg-1. This value was observed in the present study, which reported values ranging from 18357 kJ·kg-1 (IAC 301ill.) to 19070 kJ·kg-1 (IAC 311ill.). Werther et al. (2000) report an HHV for rubber tree residues of 18410 kJ·kg-1. In Brazil, values between 16500 kJ·kg-1 and 18000 kJ·kg-1 are approved for commercial energy uses. Clones with higher volatile and ash contents had lower HHV, including IAC 301ill. (83,92 %), IAC 40ill. (83,75 %), RRIM 725ill. (83,69 %), IAN 873ill. (83,67 %), Fx 2261ill. (83,65 %), PB 330ill. (83,55 %), GT1ill. (83,23 %), IAC 41ill. (83,44 %), and IAC 311ill. (82,79 %), consequently reducing the energy potential of the fuel. However, fixed carbon content is directly related to calorific power, meaning that a higher fixed carbon content implies greater resistance to thermal degradation of biomass within the burning apparatus for power generation (Arantes et al. 2013). The highest contents of fixed carbon were found for clonal progenies IAC 311ill. (15,72 %) and IAC 41ill. (15,70 %). It is said that the percentage of fixed carbon refers to the fraction of coal that burns in the solid state. Therefore, fuels with high levels of fixed carbon in both clonal progenies are preferable for steel use, owing to thermal stability and high energy power (Neves et al. 2011). Ash content should be in the range of 1 % - 3 % for good energy performance (Schoninger and Zinelli 2012). Thus, the values for this energetic characteristic are within the expected pattern for good energy yield since ash values ranged from 0,42 % (IAC 301ill.) to 1,49 % (IAC 311ill.). In Brazil, species of Eucalyptus are widely used for energy production because of their rapid growth and energetic properties (Jesus et al. 2017). However, recent studies show similarity in energetic properties between rubber tree clones. Bersh et al. (2018b) characterized Eucalyptus and reported HHV values between 13970 kJ·kg-1 and 14250 kJ·kg-1, volatile materials of 83,17 % - 86,16 %, ash content of 0,57 % - 0,60 %, and 13,27 % - 14,25 % of fixed carbon content. These values show that rubber tree clones share similar and superior energetic properties, highlighting their use for bioenergy production when compared to Eucalyptus wood. 7 Maderas. Ciencia y tecnología 2023 (25): 28, 1-16 Universidad del Bío-Bío According to Paula (2003), wood from clones has higher fiber wall thickness and length, as well as higher lignin and extractive content. Consequently, this wood is increasingly recommended for energy production for its biomass production, resulting in a greater amount of mass per unit of volume and, consequently, greater energy release capacity. Wood with high lignin content contributes to high gravimetric yield and gives greater resistance to charcoal thermal degradation since this wood has more condensed structures that degrade at higher temperatures (Castro et al. 2013, Oliveira et al. 2010). In this context, clones of higher basic densities present larger dimensions in their anatomical constitution. This characteristic is not true across the clonal spectrum as clone IAC 301ill. presented lower density and smaller fiber dimensions. The increase in wood density is followed by an increase of fiber wall thickness and length (Oliveira et al. 2010), but this was not observed in the present study. IAN 873ill. was taller and wider, but still had lower density than other clones. As lignin and extractive content increase, density increases proportionally. Thus, we find an increase in the energy yield of fixed carbon content and HHV (Dias-Junior et al. 2015). This premise was not observed for rubber tree clones IAN 873ill. and IAC 40ill. in that they presented lower wood density, even while their HHV and fixed carbon content were higher than those values in other clones. The lower wood density and HHV value of IAC 301ill. may be related to the age of the tree and anatomical characteristics of the species itself (Protásio et al. 2014). Basic density is an important property of wood and should be considered for energy use of a given biomass since that characteristic is causally related to energy production. That is, the higher the density, the greater the amount of energy stocked per unit volume (Queiroz et al. 2004). It is recommended that wood used as an energy source should present values above 0,40 kg/cm3 of wood density (Alzate et al. 2005), which is confirmed for all clones listed in Table 2. The differences found in energetic properties of wood among H. brasiliensis clones result from the chemical composition values, fiber dimensions and wood density of the biomass. Studies on other species report that HHV is mainly dependent on lignin and extractive content. High levels of lignin favor the energetic properties of fixed carbon and HHV, which can be attributed to carbon-carbon bonds between monomeric phenyl-propane units present in lignin, which hinder their decomposition, and to the higher content of carbon of this molecular component of biomass (Bufalino et al. 2012, Dietenberger and Hausburg 2016). Holocellulose is considered the most abundant component of the cell wall. However, it is amorphous and offers no resistance to high temperatures of thermal degradation, hampering its use in energy production because of its negative interference in biomass energy, HHV and physics properties (Tan and Largervist 2011). Clones with higher holocellulose levels present lower HHV, specifically IAC 326ill., Fx 2261ill., IAC 41ill., and GT1ill. Energy from biomass is usually obtained through thermochemical technologies, especially combustion. High ash content is disadvantageous because it decreases the transfer of heat in fuel and increases corrosion of the equipment used in the process. It also decreases the HHV of biomass (Protásio et al. 2014, Soares et al. 2014). Only a few studies have reported on the characteristics of wood for energy purposes. For example, Menucelli et al. (2019) studied 10 clonal progenies of rubber tree and reported characteristics superior to clones evaluated in the present study, such as fiber length between 1189 μm - 1097 μm, fiber wall thickness of 4,55 μm - 5,13 μm, extractive contents between 12,42 % - 16,34 %, lignin content between 27,51 % - 22,47 %, and wood density from 0,57 kg/cm-3 to 0,66 kg/cm-3. These wood characteristics promoted higher HHV content between 18592 kJ·kg-1 and 19757 kJ·kg-1, values that could be attributed to differences in genetic material (Soares et al. 2014). Fourier transform infrared spectroscopy (FTIR) Table 3 summarizes the main chemical and functional groups found in the wood of H. brasiliensis clones according to Tyutkova et al. (2019), being identified by the number of waves of the carboxylic groups: = C-H (902 cm-1), C-O-O (1114 cm-1), - C-H (1464 cm-1), C = O (1743 cm -1), C-H (2924 cm -1) and O-H (3370 cm -1). 8 Maderas. Ciencia y tecnología 2023 (25): 28, 1-16 Wood quality of ten clonal progenies of rubber tree..: Amorim et al. Table 3: Characteristic bands of infrared spectra (FTIR) for Hevea brasiliensis clones and their respective functional groups. One can observe that all clones have a strong absorption band at 1114 cm-1 where the lignin present in wood is located (Schuerch 1989). However, clones IAC 40, GT1, PB 330, IAC 41, RRIM 725, and IAN 873 had higher wavelengths in this spectrum. This could be explained by the chemical structure of wood because these clones have higher levels of lignin (Table 2) and, hence, higher resistance to thermal degradation of the material, as characterized by the stretch mode of the O-H combination. According to Figure 1, the group found at 1743 cm-1 can be attributed to C=O, indicating the presence of an acetyl or carboxylic acid group derived from lignin. The high proportion of these chemical groups indicates an increase in HHV (Table 1). The same behavior was reported by Schuerch (1989). It was possible to notice a strong striation between bands 1743 cm-1 and 2924 cm-1, larger than the other spectral bands reported. The bands are characterized by strong bonding between hemicellulose and lignin, which is characterized mainly by C-H, N-H and O-H that are desirable for energy production (Popescu et al. 2011). At band 3370 cm-1, the phenolic groups bonded to hydrogen formed O-H chemical groups (Pandey and Pitman 2003). This group is responsible for high degradation resistance of the wood, which is dependent on lignin content, as was possible to observe in this study. Clones with higher lignin levels present higher absorption spectra, namely IAC 40 and GT1, in the present study (Figure 1). Figure 1: Main spectra obtained for Hevea brasiliensis wood clone. 9 Maderas. Ciencia y tecnología 2023 (25): 28, 1-16 Universidad del Bío-Bío Thermogravimetric analysis (TGA) Table 4 shows averages of mass loss of different clonal progenies of Hevea brasiliensis, as a function of temperature ranges from 50 ºC to 700 ºC. Table 4: Mean values of mass loss (%) of different clonal progenies of Hevea brasiliensis as a function of temperature ranges. * Residual mass, considering the mass of wood absolutely dry (s). The first two temperature ranges, 50 ºC - 100 ºC and 100 ºC - 250 ºC, correspond to drying of wood (Vieira 2019), making an average total of 4,68 % of initial average total loss for clonal progenies of Hevea brasiliensis. Between 250 ºC and 400 ºC, an average loss in biomass of 65,11 % was verified. It can be inferred that most of this lost mass results from degradation of hemicellulose, cellulose, and volatile emissions, as well as the start of partial lignin decomposition (López-González et al. 2013). The maximum rate observed among clonal progenies of Hevea brasiliensis in this range of degradation refers to the maximum degradation of cellulose, as this constituent corresponds to 40 % - 45 % of the wood (Pereira et al. 2013). According to Figure 2, it is possible to observe the mass losses of Hevea brasiliensis clones at different temperatures. Figure 2: TG and DTG curves of mass losses for Hevea brasiliensis clones. 10 Maderas. Ciencia y tecnología 2023 (25): 28, 1-16 Wood quality of ten clonal progenies of rubber tree..: Amorim et al. Between 400 and 600 ºC, the average mass loss was 5,94 %. According to Shen et al. (2010) for this temperature range, wood thermal degradation of 5 % to 10 % can be expected, as was observed in Hevea brasiliensis. The general average mass loss observed in the 600 ºC to 700 ºC range was 1,47 %, showing that hemicelluloses and celluloses were totally degraded and that the observed mass loss refers to lignin in small proportion since it is a more thermally stable wood component. The absence of a peak of degradation related to lignin likely results from the fact that its thermal decomposition occurs over a wide temperature range, emphasizing that only a fraction decomposes at temperatures below 450 ºC, as mentioned by Huang et al. (2009). The residual mass refers to total wood that is converted into charcoal at the end of the carbonization process. Lower residual masses were observed for clones IAC 41ill. (18,45 %) and IAN 873ill. (21,95 %), but higher residual masses for IAC 326ill. (24,99 %) and IAC 301ill. (24,49 %). The values for other clonal progenies were close. These differences between residual masses can be explained by the clonal effect of the species (Vieira 2019). In Brazil, Eucalyptus clones are widely cultivated for energy production (Ferreira et al. 2017). However, it was found that clonal progenies of Hevea brasiliensis are more thermally resistant than 12 Eucalyptus clones studied by Vieira (2019), which obtained an average residual mass of 22,03 %. On the other hand, they are less thermally resistant to Eucalyptus clones studied by Pereira et al. (2013), who report residual masses at 7,5 years with values of 25,11 %, these differences being attributed to wood chemical constitution. CONCLUSIONS The results found in this work corroborate those of Eucalyptus species used in the generation of energy. However, clonal progeny IAC 311iil. presented superior characteristics with higher values of calorific value, fixed carbon and lower content of volatile material. Through the analysis of wood quality, we identified which programs cloned with lower densities presented smaller HHV and smaller fiber components, as specifically observed in clonal progeny IAC 301. FTIR showed functional groups characteristic of wood, including =C-H, C-O-O, -C-H, C=O and O-H. They are part of the chemical composition of wood and are suitable for energy production. Wood thermogravimetric analyses did not show significant variations between clonal progenies since we were able to distinguish the stages of thermal degradation, especially those associated with hemicelluloses and cellulose in the range of 250 ºC - 400 ºC. We observed satisfactory residual mass values for the conversion of wood to charcoal. In general, the ten genetic materials of Hevea brasiliensis have biomass suitable for commercial energetic use and are highly viable owing to their physical, chemical, energy and thermal characteristics. AUTHORSHIP CONTRIBUTIONS E. P. A.: Conceptualization, Resources, Data Curation, Funding aquisition, Investigation, Methodology, Validation, Writing- review & editing; E. L. L.: Conceptualization, Validation, Writing-Original draft, Data Curation, Software; M. L. M. F.s: Conceptualization, Resources; F. M. Y.i: Conceptualization, Resources; F. G. D. S. J.: Conceptualization, Resources; M. A. D. M.: Conceptualization; J. C.: Conceptualization; M. L. T. D. M.: Conceptualization; P. D. S. G.: Conceptualization 11 Maderas. Ciencia y tecnología 2023 (25): 28, 1-16 Universidad del Bío-Bío ACKNOWLEDGMENTS This study was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code- 001. 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Hindawi Publishing Corporation The Scientific World Journal Volume 2013, Article ID 725643, 8 pages http://dx.doi.org/10.1155/2013/725643 Research Article Combined Effect of Buoyancy Force and Navier Slip on MHD Flow of a Nanofluid over a Convectively Heated Vertical Porous Plate Winifred Nduku Mutuku-Njane1 and Oluwole Daniel Makinde2 1 2 Mechanical Engineering Department, Cape Peninsula University of Technology, P.O. Box 1906, Bellville 7635, South Africa Faculty of Military Science, Stellenbosch University, Private Bag X2, Saldanha 7395, South Africa Correspondence should be addressed to Winifred Nduku Mutuku-Njane; winnieronnie1@yahoo.com Received 18 May 2013; Accepted 19 June 2013 Academic Editors: E. Konstantinidis and L. Nobile Copyright © 2013 W. N. Mutuku-Njane and O. D. Makinde. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. We examine the effect of magnetic field on boundary layer flow of an incompressible electrically conducting water-based nanofluids past a convectively heated vertical porous plate with Navier slip boundary condition. A suitable similarity transformation is employed to reduce the governing partial differential equations into nonlinear ordinary differential equations, which are solved numerically by employing fourth-order Runge-Kutta with a shooting technique. Three different water-based nanofluids containing copper (Cu), aluminium oxide (Al2 O3 ), and titanium dioxide (TiO2 ) are taken into consideration. Graphical results are presented and discussed quantitatively with respect to the influence of pertinent parameters, such as solid volume fraction of nanoparticles (𝜑), magnetic field parameter (Ha), buoyancy effect (Gr), Eckert number (Ec), suction/injection parameter (𝑓𝑤 ), Biot number (Bi), and slip parameter (𝛽), on the dimensionless velocity, temperature, skin friction coefficient, and heat transfer rate. 1. Introduction Magnetohydrodynamic (MHD) boundary layer flow of an electrically conducting viscous incompressible fluid with a convective surface boundary condition is frequently encountered in many industrial and technological applications such as extrusion of plastics in the manufacture of Rayon and Nylon, the cooling of reactors, purification of crude oil, textile industry, polymer technology, and metallurgy. As a result, the simultaneous occurrence of buoyancy and magnetic field forces on fluid flow has been investigated by many researchers [1–5]. In their investigations, all the authors mentioned above assumed the no-slip boundary conditions. However, more recently, researchers have investigated the flow problem taking slip flow condition at the boundary [6–9]. On the other hand, with the advent of nanofluids, there has been wide usage of recently discovered smart fluid in many industrial and biomedical applications. Nanofluid concept is employed to designate a fluid in which nanometersized particles are suspended in conventional heat transfer base fluids to improve their thermal physical properties. Nanoparticles are made from various materials, such as metals (Cu, Ag, Au, Al, and Fe), oxide ceramics (Al2 O3 , CuO, and TiO2 ), nitride ceramics (AlN, SiN), carbide ceramics (SiC, tiC), semiconductors, carbon nanotubes, and composite materials such as alloyed nanoparticles or nanoparticle corepolymer shell composites. It is well known that conventional heat transfer fluids, such as oil, water, and ethylene glycol, in general, have poor heat transfer properties compared to those of most solids. Nanofluids have enhanced thermophysical properties such as thermal conductivity, thermal diffusivity, viscosity, and convective heat transfer coefficients compared with those of base fluids like oil or water [10]. Several authors [11–14] have conducted theoretical and experimental investigations to demonstrate that nanofluids distinctly exhibit enhanced heat transfer properties which goes up with increasing volumetric fraction of nanoparticles. Further studies on nanofluids have been currently undertaken by scientists and engineers due to their diverse technical and biomedical applications such as nanofluid coolant: electronics cooling, vehicle cooling, transformer cooling, computers 2 The Scientific World Journal Table 1: Thermophysical properties of water and nanoparticles [23, 24]. Materials Pure water Copper (Cu) Alumina (Al2 O3 ) Titania (TiO2 ) 𝜌 (kg/m3 ) 997.1 8933 3970 4250 cooling, and electronic devices cooling; medical applications: magnetic drug targeting, cancer therapy, and safer surgery by cooling; process industries; and materials and chemicals: detergency, food and drink, oil and gas, paper and printing, and textiles. According to Aziz [15], the concept of no-slip condition at the boundary layer is no longer valid for fluid flows in microelectromechanical systems and must be replaced by slip condition. The slip flow model states a proportional relationship between the tangential components of the fluid velocity at the solid surface to the shear stress on the fluidsolid interface [16]. The proportionality is called the slip length, which describes the slipperiness of the surface [7]. Many researchers studied the effect of linear momentum and nonlinear slip on the MHD boundary layer flow with heat/mass transfer of free/forced/combined convection past different geometries [17–20]. In spite of the importance of MHD related studies on boundary layer flow problems, the possibility of fluid exhibiting apparent slip phenomenon on the solid surface has received little attention. The aim of the present study is to investigate the combined effects of buoyancy, magnetic field, suction, Navier slip, and convective heating on a steady boundary layer flow over a flat surface. In the subsequent sections the boundary layer partial differential equations first transformed into a system of nonlinear ordinary differential equations before being solved numerically using a shooting method together with the fourth-order Runge-Kutta-Fehlberg integration scheme. A graphical representation of the pertinent parameters on the flow field and heat transfer characteristics is displayed and thoroughly discussed. To our best of knowledge, the investigations of the proposed problem are new, and the results have not been published before. 2. Model Formulation The steady laminar incompressible two-dimensional MHD boundary layer flow of an electrically conducting waterbased nanofluid past a convectively heated porous vertical semiinfinite flat plate under the combined effects of buoyancy forces and Navier slip is considered. The nanofluids contain three different types of nanoparticles: Cu, Al2 O3 , and TiO2 . Let the 𝑥-axis be taken along the direction of plate, and let 𝑦-axis be normal to it. The left side of the plate is assumed to be heated by convection from a hot fluid at temperature 𝑇𝑓 , which provides a heat transfer coefficient ℎ𝑓 , while the right surface is subjected to a stream of an electrically conducting cold nanofluid at temperature 𝑇∞ in the presence of a transverse magnetic field of strength 𝐵0 applied parallel to the 𝑦-axis, as shown in Figure 1. The induced magnetic field due to the motion of the electrically conducting fluid is 𝑐𝑝 (J/kgK) 4179 385 765 686.2 𝑘 (W/mK) 0.613 401 40 8.9538 𝜎 (S/m) 5.5 × 10−6 59.6 × 106 35 × 106 2.6 × 106 x g u = U∞ , T = T∞ Bo 𝜆u = 𝜇f 𝜕u 𝜆 𝜕y u  = Vw Nanofluid 𝜕T −kf = hf (Tf − T) 𝜕y  y Figure 1: Flow configuration and coordinate system. negligible. It is also assumed that the external electrical field is zero and that the electric field due to the polarization of charges is negligible (see Table 1). Assuming a Boussinesq incompressible fluid model, the continuity, momentum, and energy equations describing the flow can be written as 𝜕𝑢 𝜕V + = 0, 𝜕𝑥 𝜕𝑦 𝑢 𝜇𝑛𝑓 𝜕2 𝑢 𝑑𝑈 𝜕𝑢 𝜕𝑢 + 𝛽𝑛𝑓 𝑔 (𝑇 − 𝑇∞ ) +V = 𝑈∞ ∞ + 𝜕𝑥 𝜕𝑦 𝑑𝑥 𝜌𝑛𝑓 𝜕𝑦2 − 𝑢 𝜎𝑛𝑓 𝐵02 (𝑢 − 𝑈∞ ) 𝜌𝑛𝑓 , 𝜇𝑛𝑓 𝑘𝑛𝑓 𝜕2 𝑇 𝜕𝑇 𝜕𝑢 2 𝜕𝑇 + ( +V = ) 𝜕𝑥 𝜕𝑦 (𝜌𝑐𝑝 )𝑛𝑓 𝜕𝑦2 (𝜌𝑐𝑝 )𝑛𝑓 𝜕𝑦 + 𝜎𝑛𝑓 𝐵02 (𝜌𝑐𝑝 )𝑛𝑓 2 (𝑢 − 𝑈∞ ) . (1) The boundary conditions at the plate surface and at the free stream may be written as 𝜆𝑢 (𝑥, 0) = 𝜇𝑓 −𝑘𝑓 𝜕𝑢 (𝑥, 0) , 𝜕𝑦 V (𝑥, 0) = 𝑉𝑤 , 𝜕𝑇 (𝑥, 0) = ℎ𝑓 [𝑇𝑓 − 𝑇 (𝑥, 0)] , 𝜕𝑦 𝑢 (𝑥, ∞) = 𝑈∞ (𝑥) , 𝑇 (𝑥, ∞) = 𝑇∞ , (2) The Scientific World Journal 3 where (𝑢, V) are the velocity components of the nanofluid in the 𝑥- and 𝑦-directions, respectively, 𝑇 is the nanofluid temperature, 𝑈∞ (𝑥) = 𝑎𝑥 is the free stream velocity (which implies that the free stream fluid velocity is increasing with axial distance along the plate surface), 𝑇∞ is the free stream temperature, 𝑔 is acceleration due to gravity, 𝜆 is the slip coefficient, 𝜇𝑛𝑓 is dynamic viscosity of the nanofluid, 𝜌𝑛𝑓 is density of the nanofluid, 𝑘𝑛𝑓 is thermal conductivity of the nanofluid, 𝜎𝑛𝑓 is electrical conductivity of the nanofluid, (𝜌𝑐𝑝 )𝑛𝑓 is heat capacity at constant pressure of the nanofluid, and 𝛽𝑛𝑓 is volumetric expansion coefficient of the nanofluid which are defined as [21, 22] 𝜇𝑓 , 𝜌𝑛𝑓 = (1 − 𝜑) 𝜌𝑓 + 𝜑𝜌𝑠 , 𝜇𝑛𝑓 = 2.5 (1 − 𝜑) 𝛽𝑛𝑓 = (1 − 𝜑) 𝛽𝑓 + 𝜑𝛽𝑠 , 𝑘𝑛𝑓 𝑘𝑓 𝛼𝑛𝑓 = 𝑘𝑛𝑓 (𝜌𝑐𝑝 )𝑛𝑓 (𝑘𝑠 + 2𝑘𝑓 ) − 2𝜑 (𝑘𝑓 − 𝑘𝑠 ) = (𝑘𝑠 + 2𝑘𝑓 ) + 𝜑 (𝑘𝑓 − 𝑘𝑠 ) , + (1 − 𝜑) 2.5 2.5 (1 − 𝜑 + 𝜑𝜌𝑠 𝜑𝛽 ) (1 − 𝜑 + 𝑠 ) 𝜃 𝜌𝑓 𝛽𝑓 2.5 (1 − 𝜑 + 𝜑𝜎𝑠 ) (𝑓󸀠 − 1) = 0, 𝜎𝑓 + Gr(1 − 𝜑) − Ha(1 − 𝜑) 𝜃󸀠󸀠 + Pr 𝑘𝑓 [1 − 𝜑 + 𝜑(𝜌𝑐𝑝 ) /(𝜌𝑐𝑝 ) ] 𝑠 + Pr Ec 𝑘𝑓 𝑘𝑛𝑓 (1 − 𝜑) 1/2 𝜓 = (𝑎𝜐𝑓 ) 𝑇 − 𝑇∞ , 𝜃 (𝜂) = 𝑇𝑓 − 𝑇∞ 𝑥𝑓 (𝜂) , (4) 1/2 𝑉𝑤 = −𝑓𝑤 (𝑎𝜐𝑓 ) 2.5 (1 − 𝜑 + 2 𝜑𝜌𝑠 ) (𝑓󸀠 ) 𝜌𝑓 , 𝜆𝑢 (𝑥, 0) = 𝜇𝑓 𝜕𝑢 (𝑥, 0) , 𝜕𝑦 (7) the boundary conditions are 𝑓󸀠 (0) = 𝛽𝑓󸀠󸀠 (0) , 𝑓 (0) = 𝑓𝑤 , 𝜃󸀠 (0) = Bi [𝜃 (0) − 1] , 𝑓󸀠 (∞) = 1, (8) 𝜃 (∞) = 0, where a prime symbol denotes derivative with respect to 𝜂, 𝑓𝑤 is a constant with 𝑓𝑤 > 0 representing suction rate at the plate surface, 𝑓𝑤 < 0 corresponds to injection, 𝑓𝑤 = 0 shows an impermeable surface, 𝜆 = 0 represents highly lubricated surface, and 𝜆 = ∞ corresponds to a normal surface. The local Reynolds number (Re𝑥 ), Grashof number (Gr), Hartmann number (Ha), Prandtl number (Pr), Eckert number (Ec), slip parameter (𝛽), and Biot number (Bi), are defined as Re𝑥 = 𝑈∞ 𝑥 , 𝜐𝑓 Ha = After introducing (5) into (1) and (2), we obtain the following ordinary differential equations: − (1 − 𝜑) 𝑘𝑛𝑓 Taking into account the variable plate surface permeability and the hydrodynamic slip boundary functions defined, respectively, as where 𝜂 is the similarity variable and 𝜓 is the stream function defined as 𝜕𝜓 𝜕𝜓 , V=− . 𝑢= (5) 𝜕𝑦 𝜕𝑥 𝜑𝜌 2.5 𝑓󸀠󸀠󸀠 + (1 − 𝜑) (1 − 𝜑 + 𝑠 ) 𝑓𝑓󸀠󸀠 𝜌𝑓 Ha Pr Ec 𝑘𝑓 (6) where 𝜑 is the nanoparticle volume fraction (𝜑 = 0 correspond to a regular fluid), 𝜌𝑓 and 𝜌𝑠 are the densities of the base fluid and the nanoparticle, respectively, 𝛽𝑓 and 𝛽𝑠 are the thermal expansion coefficients of the base fluid and the nanoparticle, respectively, 𝑘𝑓 and 𝑘𝑠 are the thermal conductivities of the base fluid and the nanoparticles, respectively, (𝜌𝑐𝑝 )𝑓 and (𝜌𝑐𝑝 )𝑠 are the heat capacitance of the base fluid and the nanoparticle, respectively, and 𝜎𝑠 and 𝜎𝑓 are the electrical conductivities of the base fluid and the nanofluid, respectively. In order to simplify the mathematical analysis of the problem, we introduce the following dimensionless variables: 𝑦, (𝑓󸀠󸀠 ) + , (3) 1/2 2 2.5 𝑓𝜃󸀠 2 𝜑𝜎𝑠 ) (𝑓󸀠 − 1) = 0. 𝜎𝑓 𝜎𝑛𝑓 = (1 − 𝜑) 𝜎𝑓 + 𝜑𝜎𝑠 , 𝑎 ) 𝜐𝑓 𝑓 𝑘𝑛𝑓 × (1 − 𝜑 + (𝜌𝑐𝑝 )𝑛𝑓 = (1 − 𝜑) (𝜌𝑐𝑝 )𝑓 + 𝜑(𝜌𝑐𝑝 )𝑠 , 𝜂=( 𝜑𝜌𝑠 ) 𝜌𝑓 (1 − 𝜑 + Ec = Gr = 𝜎𝑓 𝐵𝑜 2 𝜌𝑓 𝑎 , 2 𝑈∞ 𝐶𝑝𝑓 (𝑇𝑓 − 𝑇∞ ) Bi = ℎ𝑓 𝑘𝑓 𝛽𝑓 𝑔 (𝑇𝑓 − 𝑇∞ ) 𝑈∞ 𝑎 Pr = 𝛼𝑓 , 𝜇𝑓 𝑎 𝛽= √ , 𝜆 𝜐𝑓 , √ 𝜐𝑓 , 𝜐𝑓 𝑎 . (9) 4 The Scientific World Journal The physical quantities of practical significance in this work are the skin friction coefficient 𝐶𝑓 and the local Nusselt number Nu, which are expressed as 𝜏𝑤 , 2 𝜌𝑓 𝑈∞ 𝑥𝑞𝑤 Nu = 𝑘𝑓 (𝑇𝑓 − 𝑇∞ ) , 𝜕𝑢 󵄨󵄨󵄨󵄨 󵄨 , 𝜕𝑦 󵄨󵄨󵄨𝑦=0 𝑞𝑤 = −𝑘𝑛𝑓 𝜕𝑇 󵄨󵄨󵄨󵄨 󵄨 . 𝜕𝑦 󵄨󵄨󵄨𝑦=0 0.8 (10) where 𝜏𝑤 is the skin friction and 𝑞𝑤 is the heat flux from the plate which are given by 𝜏𝑤 = 𝜇𝑛𝑓 0.9 0.7 f󳰀 (𝜂) 𝐶𝑓 = 1 0.6 0.5 (11) 0.4 Putting (11) into (10), we obtain = 2.5 (1 − 𝜑) Re−1/2 Nu = − 𝑥 Gr = Bi = Ec = 𝜑 = 𝛽 = 0.1 0.3 1 𝑘𝑛𝑓 𝑘𝑓 fw = 0.2, Ha = 10−12 󸀠󸀠 𝑓 (0) , 0.2 (12) 𝜃󸀠 (0) . The set of (6) and together with the boundary conditions (8) are coupled nonlinear boundary value problems which are solved numerically using a shooting algorithm with a Runge-Kutta Fehlberg integration scheme. This method involves transforming (6) and (8) into a set of initial value problems which contain unknown initial values that need to be determined by guessing, after which a fourth order Runge-Kutta iteration scheme is employed to integrate the set of initial valued problems until the given boundary conditions are satisfied. The entire computation procedure is implemented using a program written and carried out using Maple computer language. From the process of numerical computation, the fluid velocity, the temperature, the skin friction coefficient, and the Nusselt number are proportional to 𝑓󸀠 (𝜂), 𝜃(𝜂), 𝑓󸀠󸀠 (𝜂), and 𝜃󸀠 (𝜂), respectively. 0 1 2 3 𝜂 Cu-water Al2 O3 -water TiO2 -water Figure 2: Velocity profiles for different nanofluids. 1 0.9 0.8 0.7 f󳰀 (𝜂) Re1/2 𝑥 𝐶𝑓 0.6 0.5 3. Results and Discussion Physically realistic numerical values were assigned to the pertinent parameters in the system in order to gain an insight into the flow structure with respect to velocity, temperature, skin friction, and Nusselt’s number. The results were presented graphically in Figures 2–13, and conclusions are drawn for the flow field. The Prandtl number is kept constant at 6.2 [21]. Ha = 0 corresponds to absence of magnetic field, and 𝜑 = 0 is regular fluid. 3.1. Dimensionless Velocity Profiles. Figures 2–4 illustrate the effects of various thermophysical parameters on the nanofluids velocity profiles. Generally, it is noted that the fluid velocity increases gradually from zero at the plate surface to the free stream prescribed value far away from the plate satisfying the boundary conditions. Figure 2 shows that the momentum boundary layer thickness for Cu-water nanofluid is smaller than the rest of the nanofluids consequently, Cuwater nanofluid tends to flow closer to the convectively heated plate surface and serves as a better coolant than the other nanofluids. It is observed in Figures 3 and 4 that an increase 0.4 0.3 Gr = Bi = 𝛽 = 0.1, fw = 0.2 0.2 0 1 𝜂 2 3 𝜑 = 0, 0.02, 0.08 Ha = 10−15 , 10−12 , 10−11 Ec = 3, 5, 7 Figure 3: Velocity profiles with increasing Ha, 𝜑, and Ec. in the magnetic field intensity (Ha), nanoparticle volume fraction (𝜑), Eckert number (Ec), Grashof number (Gr), and the suction/injection parameter (𝑓𝑤 ) causes an overshoot of the fluid velocity towards the plate surface hence decreasing both the momentum boundary layer thickness and the fluid velocity. From the physics of the problem, an increase in the The Scientific World Journal 5 1 Gr = Bi = Ec = 𝜑 = 𝛽 = 0.1, fw = 0.2, Ha = 10−12 0.25 0.9 0.8 0.2 𝜃(𝜂) f󳰀 (𝜂) 0.7 0.6 0.5 0.15 0.1 0.4 0.05 0.3 𝜙 = Ec = Bi = 0.1, Ha = 10−12 0.2 0 1 𝜂 2 0 3 3.2. Dimensionless Temperature Profiles. Figures 5–7 show the effects of various parameters on the temperature profile. In general, the maximum fluid temperature is achieved at the plate surface due to the convectional heating but decreases exponentially to zero far away from the plate surface satisfying the free stream conditions. As expected, at the plate surface, Cu-water has the highest temperature and a greater thermal boundary layer thickness than the other two nanofluids, as seen in Figure 5. This is in accordance with the earlier observation, since the Cu-water nanofluid is more likely to absorb more heat from the plate surface owing to its close proximity to the hot surface. It is observed from Figure 6, that increasing Ha, 𝜑, Bi, and Ec leads to an increase in both the fluid temperature and the thermal boundary layer thickness. This can be attributed to the additional heating due resistance of fluid flow as a result of the magnetic field, the presence of the nanoparticle, the increased rate at which the heat moves from the hot fluid to the plate and the additional heating as a result of the viscous dissipation. On the other hand, it is evident that surface slipperiness and suction affect the temperature of the fluid inversely. This 1.5 2 Figure 5: Temperature profiles for different nanofluids. 0.7 𝛽 = Gr = 0.1, fw = 0.2 0.6 0.5 0.4 𝜃(𝜂) magnetic field intensity leads to an increase in the Lorentz force which is a retarding force to the transport phenomena. This retarding force can control the nanofluids velocity which is useful in numerous applications such as magneto hydrodynamic power generation and electromagnetic coating of wires and metal. We also note that the fluid velocity at the plate surface increases with an increase in the slip parameter (𝛽). This is in agreement with the fact that higher 𝛽 implies an increase in the lubrication and slipperiness of the surface. 1 𝜂 Cu-water Al2 O3 -water TiO2 -water fw = −0.4, 0, 0.3 Gr = 0, 2, 4 𝛽 = 0.5, 1, 3 Figure 4: Velocity profiles with increasing Gr, 𝛽, and 𝑓𝑤 . 0.5 0.3 0.2 0.1 0 0 0.5 1 𝜂 𝜑 = 0, 0.01, 0.03 Ha = 10−15 , 10−12 , 10−11 1.5 2 Bi = 0, 0.3, 1 Ec = 0.2, 0.215, 0.3 Figure 6: Temperature profiles with increasing 𝜑, Ha, Bi, and Ec. is clearly seen from Figure 7, where both temperature and thermal boundary layer decrease as 𝑓𝑤 and 𝛽 increase. 3.3. Effects of Parameters Variation on the Skin Friction and Nusselt Number. Figures 8–13 demonstrate the effects of the various pertinent parameters at the plate surface for both the skin friction coefficient and the local Nusselt number 6 The Scientific World Journal 𝜑 = Gr = Ec = Bi = 0.1, Ha = 10−12 3.5 0.3 √Rex Cf 3 𝜃(𝜂) 0.2 2.5 2 0.1 1.5 𝛽 = Bi = Gr = 0.1, fw = 0.2 0 0 0.5 1 𝜂 1.5 2 0 fw = −0.01, 0.1, 0.2 𝛽 = 0.01, 0.2, 0.3 0.05 0.1 𝜑 0.15 0.2 Ha = 10−15 , 10−13 , 10−12 Gr = 1, 3, 4 Ec = 0.5, 2, 3 Figure 7: Temperature profiles with increasing 𝛽, Gr, and 𝑓𝑤 . Figure 9: Effects of increasing Gr, Ha, and Ec on local skin friction. 3 Ec = 𝛽 = Gr = Bi = 0.1, fw = 0.2, Ha = 10−12 2.8 2.6 2.6 2.2 2.4 2 2.2 √Rex Cf √Rex Cf 𝜑 = Ec = Bi = Gr = 0.1, Ha = 10−12 2.4 2 1.8 fw = −0.4, 0.1, 0.4 1.8 1.6 1.4 1.6 1.2 1.4 1 0.8 0 0.05 0.1 𝜙 0.15 0.2 Cu-water Al2 O3 -water TiO2 -water 0 0.5 1 1.5 𝛽 Figure 10: Effects of increasing 𝛽 and 𝑓𝑤 on local skin friction. Figure 8: Local skin friction profiles for different nanofluids. (rate of heat transfer). The presence of nanoparticle in the convectional fluid leads to an increase in the skin friction, as seen in Figure 8, where increasing the nanoparticle volume fraction increases the skin friction for the three nanoparticles (Cu, Al2 O3 , and TiO2 ) used, with Cu-water exhibiting the highest increment. This is as expected, since Cu-water moves closer to the plate surface leading to an elevation in the velocity gradient at the plate surface. As expected, increasing Ha, Gr, Ec, and 𝑓𝑤 leads to an increase in the skin friction coefficient, while an increase in 𝛽 reduces the skin friction coefficient as shown in Figures 9 and 10. There is an increase in the rate of heat transfer with an increase in 𝜑, Bi, and 𝑓𝑤 as seen in Figures 11-12, with Al2 O3 exhibiting the highest increment. The converse is seen with increasing Ha as shown in Figure 13. The Scientific World Journal 7 0.12 Ec = 𝛽 = Gr = Bi = 0.1, fw = 0.2, Ha = 10 0.12 −12 𝜙 = 𝛽 = Gr = Bi = 0.1, fw = 0.2 0.1 Ha = 10−15 , 10−13 , 10−12 0.08 Nu/√Rex Nu/√Rex 0.11 0.1 0.06 0.04 0.02 0.09 0 0.1 −0.02 0 0.05 0.1 𝜑 0.15 0.2 Al2 O3 -water Cu-water TiO2 -water Figure 11: Local Nusselt number for different nanofluids. Ec = 𝛽 = Gr = 0.1, Ha = 10−12 0.14 0.13 0.12 Nu/√Rex 0.11 0.1 0.09 0.08 0.2 0.3 Ec 0.4 0.5 Figure 13: Effects of increasing 𝜑, Ha, and Ec on local Nusselt number. nonlinear partial differential equations were transformed into a self-similar form and numerically solved using shooting technique with a fourth-order Runge-Kutta-Fehlberg integration scheme, putting into consideration the enhanced electrical conductivity of the convectional base fluid due to the presence of the nanoparticles. Our results showed that the fluid velocity increases, while the local skin friction decreases with the increase in the slip parameter (𝛽), but the reverse is observed with the increase in the magnetic field intensity (Ha), nanoparticle volume fraction (𝜑), Eckert number (Ec), Grashof number (Gr), and the suction/injection parameter (𝑓𝑤 ). Both the temperature and the thermal boundary layer thickness are enhanced by increasing the magnetic field intensity (Ha),nanoparticle volume fraction (𝜑), Eckert number (Ec), and the intensity of Newtonian heating (Bi), while the cooling effect on the convectively heated plate surface is enhanced by increasing the velocity slip (𝛽) and suction parameter (𝑓𝑤 ). 0.07 Acknowledgments 0.06 0 0.05 0.1 𝜑 0.15 0.2 Bi = 0.07, 0.1, 0.12 fw = −0.1, 0.01, 0.3 Figure 12: Effects of increasing 𝜑, Bi, and Ha on local Nusselt number. 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Mahdy, “Natural convection flow and heat transfer enhancement of a nanofluid past a truncated cone with magnetic field effect,” World Journal of Mechanics, vol. 2, pp. 272–279, 2012. International Journal of Rotating Machinery Engineering Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Distributed Sensor Networks Journal of Sensors Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Control Science and Engineering Advances in Civil Engineering Hindawi Publishing Corporation http://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Volume 2014 Submit your manuscripts at http://www.hindawi.com Journal of Journal of Electrical and Computer Engineering Robotics Hindawi Publishing Corporation http://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Volume 2014 VLSI Design Advances in OptoElectronics International Journal of Navigation and Observation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Chemical Engineering Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Volume 2014 Active and Passive Electronic Components Antennas and Propagation Hindawi Publishing Corporation http://www.hindawi.com Aerospace Engineering Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Volume 2014 International Journal of International Journal of International Journal of Modelling & Simulation in Engineering Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Shock and Vibration Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Advances in Acoustics and Vibration Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 

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ACTA IMEKO ISSN: 2221-870X December 2020, Volume 9, Number 5, 247 - 249 METROLOGICAL RESEARCHES OF NATIONAL PRIMARY STANDARD MACHINE FOR LEEB HARDNESS SCALES A. Aslanyan1,2, E. Aslanyan1, S. Gavrilkin1, A. Shchipunov1 1 VNIIFTRI, Mendeleevo, Moscow region, Russian Federation, 2 Andrey_aslanyan@vniiftri.ru Abstract: The paper describes the study of metrological characteristics of the National primary standard machine for Leeb hardness scales. The data on the contribution of various values to the extended uncertainty of hardness measurements are presented. Keywords: Leeb hardness; National primary standard machine; Expanded uncertainty 1. INTRODUCTION From 2016 to 2018, VNIIFTRI improved the National primary standard machine for Shore D hardness scales for metals [1]. It was improved by introducing into the standard machine devices that measure the hardness of metals on the Leeb D and G scales. The modernization of the standard was necessary to ensure the traceability of hardness testers that implement the Leeb method to the National primary standard of hardness. The paper presents the values of the main components of the expanded uncertainty of measurements on the National primary standard machine for Leeb hardness scales. 2. DESCRIPTION OF THE WORK The process of reproducing the hardness numbers on the Leeb scales consists in the fall of a impact body with a diamond, ceramic or tungsten tip on a sample or hardness test block and in determining the ratio between the rebound and impact velocity of the impact body [2]. The hardness numbers on the Leeb scales are determined by the formula 𝐻𝐿 = 1000 𝑣𝑟 𝑣𝑖 (1) where 𝑣𝑟 is a rebound velocity of impact body; 𝑣𝑖 is a impact velocity of impact body. The mass of impact body, radii of the tip of impact body, impact velocity, material of the tip are standardized for each Leeb scale. The most common Leeb scales in industry are Leeb D and Leeb G scales. Requirements for these scales are presented in Table ACTA IMEKO | www.imeko.org 1, where r is a radius of the tip of impact body, m is a mass of impact body. Table 1: Requirements for Leeb scales D and G Requirements m, g r, mm 𝑣𝑖 , m/s HLD 5.45 ± 0.03 1.500±0.003 2.0500±0.0025 HLG 20.00±0.03 2.500±0.003 3.000±0.005 Devices for Leeb hardness measuring consists of tubes with electromagnetic coils, impact bodies with built-in magnets, a mechanical device for resetting impact bodies. After faulting the impack body and its passage through the coil, an electromotive force (EMF) is induced in it, which is fixed by an electronic unit. The ratio of maximum voltage after impact and before it is equal to the ratio of the impact velocity to the rebound velocity of the impact body. The electronic unit recalculates this ratio into hardness numbers on Leeb scale. The expanded uncertainty of measurement of hardness values on the Leeb scales affected by deviations from the nominal values of mass and radius of the tip of impact body, the impact velocity of impact body, the uncertainty of the measurement of the ratio of velocity at the time of rebound of the impact body at the moment of impact. When improving the primary standard machine, the traceability of all values that affect the reproduction of hardness numbers to the national primary machines is ensured: units of mass – kilogram, units of length-meter, units of time – second, units of voltage – volt. The masses of the impact bodies were measured using weights, and the radii of the tips of the impact bodies were measured using a profilometer. The impact velocity was measured using a laser triangulation displacement sensor. The displacement sensor was attached to the reference tube in a special holder. The impact body fell down the tube, the sensor, depending on the displacement of the impact body, issued an electrical signal with a frequency of 400 kHz. The time intervals during which the impact body passed the measured displacement were measured by an December 2020 | Volume 9 | Number 5 | 247 oscilloscope. The oscilloscope also measured the electrical voltage on the sensor, which was converted into displacement using the calibration function. The resulting dependence of the displacement of the impact body on time determines the velocity of the fall of the impact body. In parallel, the oscilloscope measured the voltage on the electromagnetic coil through which the impact body flew. The moment of impact of the impact body on the hardness test block was determined using an oscilloscope at the point of transition of the "Voltage - time" curve in a straight line when the impact body flew through a tube with a coil. The standard uncertainty of measurement of displacement by the sensor was determined from the "Voltage-displacement" calibration curve. The velocity of an impact body with a magnet passaging through an electromagnetic coil is proportional to the voltage induced on the coil. The ratio of the impact body velocity when it is impact to the impact body velocity when it is rebound is equal to the ratio of voltages at these times. The electrical voltages generated on the coil after passage through it the sides with the magnet were measured using an oscilloscope connected to the coil in parallel with the electronic unit. The sensitivity coefficients of the influencing quantities included in equation (1) were determined by differentiating equation (1) by the influencing quantity. The sensitivity coefficients of values not included in equation (1) were found experimentally. Among the values not included in equation (1) are the radius of rounding of the impact body and the mass of the impact body. The sensitivity coefficients for these values were determined by a small change in only one parameter and determining the change in hardness. The sensitivity coefficient is equal to the ratio of the change in hardness to the change in this parameter. Table 2 shows the values that affect measurements. Table 3 shows the values that affect measurements. Table 2: The sensitivity coefficients of influence quantities The standard combined uncertainty of measurement on Leeb hardness scale HLD is no more than 1.5. The expanded uncertainty of measurements on the Leeb HLD scale with a coverage factor of 2 is no more than 3.0. The standard combined uncertainty of measurement on Leeb hardness scale HLG is no more than 1.39, and the expanded uncertainty of measurements on the HLG scale with a coverage factor of 2 is no more than 2.8. Influencing values Mass of impact body Radii of the tip of impact body Impact velocity Hardness level, HLD 466 601 765 466 601 765 466 601 765 The ratio of the impact body velocity to the impact body velocity ACTA IMEKO | www.imeko.org All range Sensitive coefficients -21.3 HLD/g -18.7 HLD/g -9.4 HLD/g 314.1 HLD/mm 286.3 HLD/mm 201.5 HLD/mm -227.3 HLD·s/m -293.2 HLD·s/m -372.7 HLD·s/m 1000 HLD sensitivity coefficients for the Leeb D hardness sensitivity coefficients for the Leeb G hardness Table 3: The sensitivity coefficients of influence quantities Influencing values Hardness level, HLG Mass of impact body 426 560 636 Radii of the tip of impact 426 body 560 636 Impact velocity 426 560 636 The ratio of the impact body velocity to the All range impact body velocity Sensitive coefficients -19.5 HLG/g -14.3 HLG/g -7.9 HLG/g 293.1 HLG/mm 245.3 HLG/mm 189 HLG/mm -142 HLG·s/m -186.7 HLG·s/m -211.7 HLG·s/m 1000 HLG Table 4 shows the standard measurement uncertainty values that affect the reproduction of hardness numbers on the scales HLD, HLG. Table 4: The contribution of influencing values to the standard combined uncertainty of hardness measurements on the Leeb scales Influencing values Mass of impact body Radii of the tip of impact body Impact velocity The ratio of the impact body velocity to the impact body velocity HLD 0.06 0.12 0.75 HLG 0.09 0.09 0.64 1.29 1.22 3. SUMMARY Metrological researches of the National primary standard machine allowed us to estimate the expanded uncertainty of measurements on the Leeb HLD and HLG hardness scales. December 2020 | Volume 9 | Number 5 | 248 4. REFERENCES [1] E. G. Aslanyan, M. V. Balakhanov, N. S. Gusyatinskaya, A. S. Doinikov, V. I. Kozlov, N. A. Safarov, V. R. Shlegel, Measurement Techniques, vol. 45, no. 3, pp. 223–227, 2002. ACTA IMEKO | www.imeko.org [2] K. Herrmann, Hardness Testing Principles and Applications. Leeb Method, Published by ASM International, Novelty, USA, pp. 78–86, 2011. December 2020 | Volume 9 | Number 5 | 249 

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JOURNAL OF THE NEUROLOGICAL SCIENCES 13 Atheroma of the Larger Arteries of the Brain in a Polish Population: A Study of 600 Cases M. J. MOSSAKOWSKI, Z. KRAŚNICKA and L. IWANOWSKI The Institute of Neuropathology of the Polish Academy of Sciences, Warsaw (Poland) INTRODUCTION In 1959 the World Federation of Neurology initiated a series of epidemiological studies in cerebro-vascular disease, with the purpose of establishing the importance of various factors, including geographic, climatic, racial, nutritional and occupational, upon the spread of degenerative arterial disease of the brain, as well as on its course, frequency, intensity and distribution. Several papers on the subject have already been published (Baker and Iannone 1959a-c, 1961; Baker et al. 1960, 1961). These studies were carried out on material from two different centres, namely the United States and Norway. In Poland, epidemiological studies on brain atheroma, carried out according to WFN standards, were started in 1960. They were made upon case­ material from Warsaw, a large administrative, cultural and partly industrial centre. Studies going on at present are being carried out in two other regions of Poland - in the neighbourhood of Lublin, a rural part of the country, and in Bydgoszcz, a large in­ dustrial centre. The material, made up of data from these three centres, should be re­ presentative of Poland. Data previously collected have been published in several papers in which more detailed questions were discussed (Majdecki and Zelman 1961; Dambska, Kraśnicka, Mandybur, Wiśniewski, in the press). The aim of the present work is to analyze the frequency and intensity of atheroma­ tous changes in the larger arteries of the brain in the Polish population along the lines of the work done by Baker and Iannone (I959a-c) and by Baker et al. (I960) on Norwegian and American material. MATERIAL AND METHOD The studies were carried out on 600 unselected patients who had died in the State University Hospital No. 1 in Warsaw. As a postmortem examination is performed as a routine on almost all cases in state hospitals, our material can be considered as being representative of the average population in this region of Poland. In our material, the first decades of life are represented by only a few cases, because there is a separate Pediatric University Hospital with its own autopsy service. J. neurol. Sci. (1964) 1: 13-23 http://rcin.org.pl 14 M. J. MOSSAKOWSKI, Z. KRASNICKA, I . 1WANOWSKI The pathological studies were carried out according to the standards laid down by the World Federation of Neurology (1959). The atheromatous changes in the larger arteries of the brain were coded in the following manner, ranging: (1) An opacity to a very thin plaque involving only a small sector of the circum­ ference of a vessel: no narrowing of the lumen; (2) A thin plaque involving more than half the circumference of the vessel with minimal narrowing of the lumen; or a small thick plaque causing less than 25% narrowing of the lumen; (3) A thick plaque involving the entire circumference with narrowing of the lumen; or a localized plaque causing 25-50% narrowing; (4) A very thick plaque involving the entire circumference with moderate or marked narrowing of the lumen (pipestem); or a localized plaque, producing more than 50% narrowing of the lumen. Following the practice of Baker et al. (1960) we divided all our cases into several groups, depending on the degree of advancement of the atheromatous changes. Group ‘O’ included cases with no atheromatous changes. The sum of the changes in each case, classified according to the WFN coding system, was the basis for grouping cases with atheromatous changes. Those in which the general ‘sum of atheromatosis’ was less than 10 were classified as mild cases; those with ‘a sum of atheromatosis' between 11 and 20 as moderate; and those with more than 21 as severe. We accepted this scheme of classification despite certain reservations as to the method because of the mechanics and subjectivity of evaluation. The principal reservation lay in the fact that the limits of the groups with severe atheromatosis were too wide, and might include cases from 21 to 112 points. It was true that none of our cases reached the maximal number of points, but it still seemed to us that there was a great difference, from both the clinical and morphological points of view, between cases with 21 and those scoring 70 points: the latter were not rare in our material. In order to avoid this inaccuracy, Majdecki and Zelman (1961) introduced an additional group to include cases in which the sum of ‘atheromatous points’ was higher than 40, and they called this, the group with the most severe atheromatosis. However, in order to make possible the comparison of our material with data obtained from American and Norwegian populations, we have retained Baker’s criteria of classification. The series was made up of almost the same number of males and females (303 women and 297 men). The ages of patients varied from 11-99 years, but the cases in the sixth, seventh and eighth decades of life were more numerous. Under 20 and over 90 years of age, there were only single cases. RESULTS Atheromatous changes were present in 68.7% of all cases. Mild atheroma was present in 27.3%, moderate in 11.5%, and severe in 29.8%; 31.3% were devoid of any atheromatous changes. Table 1 and Fig. 1 show the frequency of atheromatous changes in the various age groups. Cases with the earliest atheromatous changes in the large brain arteries were observed in the third decade of life. In these, the degen­ erative changes in the arterial walls were of mild degree. In two cases the atheroma J. neurol. Sci. (1964) 1: 13-23 http://rcin.org.pl 1 TABLE 15 RELATIONSHIP OF AGE AND SEX TO THE INTENSITY OF CEREBRAL ATHEROSCLEROSIS ATHEROMA OF THE LARGER ARTERIES OF THE BRAIN IN A POLISH POPULATION J. neurol. Sci. (1964) 1: 13-23 http://rcin.org.pl 16 M. J. MOSSAKOWSK1, Z. KRASNICKA, L. IWANOWSKI 31-40 years 41-50 years 51-60 years no atherosclerosis mild atherosclerosis moderate atherosclerosis severe atherosclerosis 61-70 years 71-80years 81-90 years Fig. 1. Frequency of cerebral atherosclerosis in the various age-groups. occurred in male patients with subacute bacterial endocarditis, aged respectively 23 and 30 years. The third in this group was a 29-year-old male, who died of cerebral hemorrhage of unknown causation. Severe atheroma in our first cases occurred in the fourth decade of life. In one this was noted in a 35-year-old male with a long history of chronic glomerulonephritis, and in the second in a 39-year-old male with a 10-year history of arterial hypertension complicated by myocardial infarction and apoplexy. Although atheromatous changes were noted in cases as early as in the fourth and fifth decades of life, these were in the great minority as compared with cases with no atheromatosis. In the group of patients in their sixties, atheromatous changes were found in more than two-thirds. At the same time there was an evident shift of the spectrum of atheromatous change from mild to severe grades. In the ninth decade only 2.94% of cases were free from degenerative arterial disease, and more than 55% were in the group with severe atheroma. In two cases in the tenth decade the presence of atheromatous changes was clear, but in both of these the changes were of moderate degree. Fig. 1 shows that the number of cases with no atheroma decreased progres­ sively with age, while the number of cases with severe atheroma steadily increased. It seemed of interest to study the frequency and severity of atheromatous changes in each of the larger cerebral arteries. Table 2 shows the frequency and degree of the atheroma within the wall of each individual component of the circle of Willis. This indicates that the internal carotids, both proximal to and at the point of trifurcation, are the most often involved by atheroma. The next commonest site is the proximal J. neuro!. Sei. (1964) 1: 13-23 http://rcin.org.pl ATHEROMA OF THE LARGER ARTERIES OF THE BRAIN IN A POLISH POPULATION 17 TABLE 2 FREQUENCY AND INTENSITY OF ATHEROMATOUS CHANGES IN LARGER CEREBRAL ARTERIES Cerebral arteries 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. commun. anter. A. basil, (p. ant.) A. basil, (p. media) A. basil, (p. post.) A. cerebri ant. dext. (p. prox.) A. cerebri ant. sin. (p. prox.) A. cerebri ant. dext. (p. dist.) A. cerebri ant. sin. (p. dist.) A. car. int. dext. (usque ad trifurc.) A. car. int. sin. (usque ad trifurc.) A. car. int. dext. (in trifurc.) A. car. int. sin. (in trifurc.) A. cerebri med. dext. (p. prox.) A. cerebri med. sin. (p. prox.) A. cerebri med. dext. (p. dist.) A. cerebri med. sin. (p. dist.) A. commun. post. dext. A. commun. post. sin. A. cerebri post. dext. (p. prox.) A. cerebri post. sin. (p. prox.) A. cerebri post. dext. (p. dist.) A. cerebri post. sin. (p. dist). A. cerebelli sup. dext. A. cerebelli sup. sin. A. vertebr. dext. A. vertebr. sin. A. cerebelli infer, post. dext. A. cerebelli infer, post. sin. Grade 1 Grade 2 Grade 3 Grade 4 Total 59 133 112 118 116 120 85 83 124 126 134 134 139 131 140 142 70 79 121 125 125 114 84 69 126 125 69 63 22 102 74 86 41 37 32 39 126 125 131 127 108 109 72 76 15 19 72 69 53 47 24 24 70 69 19 20 4 37 38 50 13 12 5 3 48 48 50 52 32 36 15 11 3 2 13 14 16 3 2 2 30 30 5 4 4 4 3 4 4 3 3 2 3 1 1 1 1 1 1 3 1 85 276 228 257 170 169 122 125 302 303 318 316 281 279 228 230 89 101 207 208 194 164 110 95 227 227 93 88 portion of the middle cerebral arteries, and the anterior and posterior portions of the basilar artery. The anterior communicating, the posterior communicating, and the various cerebellar arteries are the ones least often involved. As a rule, the symmetrical homonymous arteries are affected with the same frequency. The posterior communi­ cating arteries and the superior cerebellar arteries are the only exceptions, the left posterior communicating artery and the right superior cerebellar artery being more commonly involved. Fig. 2 illustrates the severity of atheroma in the various large cerebral arteries. The degree of severity was expressed by multiplying the frequency of involvement by the severity of involvement (in pluses according to the WFN coding system). In comparing the data shown in Table 2 and Fig. 2, one sees that the degree of intensity of atheroma in our material is identical with the frequency of involvement. The arteries most often involved are at the same time the ones which are most severely affected. Thus the most advanced atheroma is found in the internal carotid arteries; in the proximal portion of the middle cerebral arteries; and in the basilar artery (posteri­ or and anterior portions). The least advanced degree of atheroma is seen in the anteri­ or and posterior communicating arteries, and in both cerebellar arteries. We tried to determine in which of the arteries, atheromatous changes appeared J. neurol. Sci. (1964) 1: 13-23 http://rcin.org.pl 18 M. J. MOSSAKOWSKl, Z. KRASNICKA, L. 1WANOWSKI Fig. 2. Distribution of the atheromatous changes in the larger arteries of the circle of Willis. I: arteries not examined; 2: slight atherosclerosis; 3: moderate atherosclerosis; 4: severe atherosclerosis; 5: the most severe atherosclerosis. TABLE 3 FREQUENCY OF CEREBRAL ATHEROSCLEROSIS ACCORDING TO THE VARIOUS AGE-GROUPS Artery Anterior commun. Anterior basilar Middle basilar Posterior basilar Anterior cerebral prox. Anterior cerebral dist. Int. carotid before trifurc. Int. carotid at trifurc. Middle cerebral prox. Middle cerebral dist. Posterior communic. Posterior cerebral prox. Posterior cerebral dist. Superior cerebellar Vertebral Posterior inf. cerebell. 31-40 41-50 1.9 11.8 9.8 11.8 2.9 1.9 10.8 11.8 7.8 8.8 1.9 4.9 3.9 3.9 6.9 4.9 _ 10.34 8.6 12.1 5.2 1.7 17.2 14.7 9.4 8.6 2.6 3.4 5.2 1.7 10.3 1.7 Age grot.ips * (%) 51-60 61-70 11.0 40.7 33.8 37.9 21.4 16.2 44.1 44.8 40.7 30.7 5.5 27.6 19.7 14.1 29.0 13.4 13.2 60.2 47.0 51.9 36.4 27.1 64.7 67.2 55.4 40.1 10.2 47.3 35.8 21.1 48.8 18.7 71-80 81-90 27.9 63.9 52.5 61.5 43.4 32.4 72.9 75.8 72.2 65.2 4.9 50.4 41.8 25.0 54.5 21.3 38.2 79.4 73.5 64.7 52.9 38.2 77.9 86.7 82.4 73.5 5.9 72.0 61.8 22.1 61.8 29.4 * Age-groups between 21-30 and 91-100 are excluded because of the very small number of cases in each of those. J. neurol. Sci. (1964) 1: 13-23 http://rcin.org.pl ATHEROMA OF THE LARGER ARTERIES OF THE BRAIN IN A POLISH POPULATION 19 first. The slight changes observed in single cases in the third decade of life were to be seen first in the internal carotid arteries at the site of trifurcation, in the proximal portion of the middle cerebral arteries, and in the middle third of the basilar artery. Table 3 shows in what percentage of cases the individual cerebral arteries are involved according to the various age groups. This table indicates clearly that the highest percentage of atheroma in the youngest group considered (i.e. the fourth decade), occurs subsequently in the site of trifurcation of the internal carotid arteries, in the anterior and posterior portion of the basilar artery, the pre-trifurcational portion of the internal carotid, and the middle third of the basilar artery. % Group with mild atherosclerosis | |Group with moderate atherosclerosis Group with severe atherosclerosis Fig. 3. Relative frequency of atheromatous changes in the larger arteries of the circle of Willis in various groups of cerebral atherosclerosis. The next question studied was how often atheromatous changes occurred in the individual brain arteries of various groups of atheroma. In the group with severe atheroma all the larger cerebral arteries were affected in over 50% of cases. In the group with mild atheroma only, internal carotid arteries at the site of trifurcation were involved in 50%. Numerous large cerebral arteries in this group were involved in single cases only. Anterior and posterior communicating arteries and the cerebellar arteries were involved least frequently. This indicates that in cases with severe athero­ ma the pathological process was more diffuse and generalized, involving all cerebral arteries; while in the group with mild atheroma the lesions were circumscribed and limited to the large arteries which were involved most often, most severely and earliest. Table 1 presents the data concerning the frequency and severity of atheromatous changes in various age groups, and also takes into account the sex factor. The table shows some quantitative differences in both sexes, particularly in the groups with mild and severe atheroma. There is a preponderance of men in the first group, while women make up the majority in the second. J. neuro!. Sci. ( 1964) 1: 13-23 http://rcin.org.pl 20 M. J. MOSSAKOWSKI, Z. KRASNICKA, L. IWANOWSKI DISCUSSION It is of interest to compare our results obtained with Polish subjects with the obser­ vations made by Baker and Iannone (1959a—c) in a Minnesota population and also with those of Baker, Refsum and Dahl (1960). The opportunity for comparative studies was specially good as almost the same number of cases were involved, and an identical coding method was used. However, an important difference lay in the fact that our series did not contain examples from the first two decades of life which were included in the American and Norwegian studies. In general, our results are very similar to those obtained by Baker et al. (1960). The total number of cases with atheromatous changes in the largercerebral arteries was markedly higher in our series (68.7%) than in the American and Norwegian researches (54.2%,), slightly higher than this in Baker’s and Iannone’s series (1959a—c) (60%) and lower than in those de­ scribed by Baker, Refsum and Dahl(1960) (72.33%). Taking into account the child­ hood and juvenile groups in the series of Baker et al. as well as all of the material (both American and Norwegian), one would expect that the proportions would be less when Polish and combined American and Norwegian subjects are compared, and greater between Polish and Norwegian populations. Quantitative comparisons of the various atheroma groups were possible only with the Norwegian material. The percentage of cases with mild (26.5%) and severe (28.5%) atheroma was, in Baker’s material, slightly lower than in ours. The only obvious difference found was in the group with moderate atheroma, which in our material was present in 11.5% and in Baker's in 17.3%. The first mild atheromatous changes in our subjects occurred one and a half decades earlier than in the Norwegian group and one decade later than in the Minne­ sota series. The first cases with severe atheroma were noted by us in the same decade as in Baker’s material from Minnesota, but half a decade earlier than in those from Norway. With increasing age, the frequency and severity of atheromatous changes showed only slight differences between the Polish and Norwegian populations. In the seventh decade of life the number of cases with severe atheroma was 36.74% in the Polish group and only 27.1% in the Norwegian, but the number of cases with moderate atheroma in Norway was 10",, higher than in Poland. The number of cases with mild atheroma in that age-group was almost identical in both populations. In the eighth decade the differences discussed above in groups with moderate and severe atheroma were decreasing but, on the other hand, a minimal difference with slight preponder­ ance of Norwegians appeared in the group with mild atheroma. The number of cases with no atheroma in this age group was less in Baker’s material than in ours. In the ninth decade the number of cases without atheroma was only insignificantly higher in both populations, namely 4 and 2%. A much higher percentage with no atheroma in the group over 80 years was found in both the Norwegian (12%) and the American (7%) material (Baker, Iannone and Kinnard 1960). As far as the distribution of atheromatous changes in the larger brain arteries is concerned, our data are very similar to those of Baker, Refsum and Dahl (1960) among Norwegians. The principal difference is in the lesser involvement of proximal J. lieuro ISei. (1964) 1: 13-23 http://rcin.org.pl ATHEROMA OF THE LARGER ARTERIES OF THE BRAIN IN A POLISH POPULATION 21 portions of the posterior cerebral arteries and in the greater symmetry of atheromatous changes in our cases. The differences in the intensity and frequency of atheroma in the symmetrical homonymous arteries were, in our series, so slight that in no case were homonymous arteries classified among different atheromatous groups. When our data concerning the distribution of the degenerative lesions in the larger cerebral arteries are compared with those of Baker et al. (1959a-c), one finds more severe involvement of the internal carotid arteries (at the site of bifurcation), and less damage of the proximal portions of the posterior cerebral arteries in our material than in both the American and Norwegian groups. However, the severity of atheromatous involve­ ment of the larger cerebral arteries was much greater in our material than in that from Minnesota. When the intensity of atheromatous changes within individual brain arteries is compared (expressed in plus signs according to the WFN coding system) the data from our material and Baker’s were very similar except that there were but few cases rated as 4 pluses in our cases. This might mean that atheromatous changes leading to marked narrowing of the calibre of larger arteries of the brain, were rarer in the Polish than in the Norwegian population. Like Baker, Iannone and Kinnard (1960) in their studies on both American and Norwegian populations, we found no significant differences in frequency and intensity of atheroma as regards sex. Quantitative differences in the number of male and female cases in groups with mild and severe atheromatosis can be explained by the differing numbers of males and females in the age-groups concerned, especially the preponder­ ance of women among the older age-groups; for instance, in the group of 170 patients over 70 years of age, 103 were women. In our study of the time of the first appearance of atheromatous lesions, we were able to establish that the earliest changes also occurred in those arteries which were found to be involved most often and most severely. In cases in which the atheroma was less wide-spread and less advanced, irrespective of the patient's age, atheromatous changes were limited to the internal carotid, middle cerebral and basilar (anterior and posterior portion) arteries. When atheroma was intense and wide-spread, the atheromatous changes also involved other portions of the arterial tree at the base of the brain. The dependency on age of distribution of atheromatous changes seemed to be secondary. It existed inasmuch as there was an unquestionable age-factor in the generalization and intensity of atheroma. Our observations agree with the conclusions of Baker et al. (1959a-c, I960,1961) as to the relationship between the size of the vessels and the frequency and severity of atheroma. However, it seemed to us that the problem was not the size of the artery but other factors, perhaps, closely related to the blood vessel’s calibre. In this connex­ ion we had in mind the nutrition of the blood-vessel wall, (Ramsey 1936/37), as well as the metabolism and biochemistry of the arterial tissue (Kirk 1951). We were able to establish some measure of predilection in the distribution of atheromatous changes for the sites of arterial branching (Adams and Van der Eecken 1953),thusconfirming a well-known view on the role of haemodynamic traumainthepathogenesis of athero­ matous degeneration. J. neurol. Sci. (1964) 1: 13-23 http://rcin.org.pl 22 M. J. MOSSAKOWSKI, Z. KRAŚNICKA, L. IWANOWSKI SUMMARY (1) Studies were carried out upon a series of 600 unselected patients between the age­ limits of 11-99 who died in the first State University Hospital in Warsaw. (2) In 31.3% of cases there were no atheromatous changes in the larger cerebral arteries; in the remaining 68.7% of the subjects examined there were degenerative vascular changes which were classified as mild atheroma in 29.8%, in 11.5% as mo­ derate. and in 27.3% as severe. (3) Atheromatous changes were first found in cases in the third decade of life. After that age the number of cases with brain atheroma was found to increase gradually according to the patient's age. At the same time there was a definite shift within the spectrum of atheroma from changes classified as mild to those of severe degree. (4) The internal carotid arteries, the anterior and posterior portions of the basilar artery, and the middle cerebral arteries were the ones most severely involved. The degenerative atheromatous changes occurred in these situations most often and most early. In cases with mild atheroma there was clear-cut selectivity as to localization of atheromatous changes for the above-mentioned arteries; as the intensity of athero­ ma increased, the degenerative changes were also seen to occur in other regions of the cerebral arterial tree. (5) There was no great difference in the frequency and intensity of atheromatous changes in the brain arteries, with regard to sex. (6) A comparison was made with the results obtained and the data presented by Baker el al. in their American and Norwegian series. REFERENCES Adams, R. D. and H. M. Van der Eecken, (1953) Vascular diseases of the brain, Ann. Rev. Med., 4: 213. Baker A. B. and A. Iannone, (1959a) Cerebro-vascular disease, Part 1 (The large arteries of the circle of Willis), Neurology (Minneap.), 9: 321. Baker, A. B. and A. Iannone, (1959b) Cerebro-vascular disease. Part 2 (The smaller intracerebral arteries). Neurology (Minneap.), 9: 391. Baker, A. B. and A. Iannone, (1959c) Cerebro-vascular disease. Part 3 (The intracerebral arterioles), Neurology (Minneap.). 9: 441. Baker. A. B. and A. Iannone, (1961 (Cerebro-vascular disease, Part7(Astudy of etiological mechan­ isms), Neurology (Minneap.), II : 23. Baker, A. B.. A. Iannone and J. Kinnard, (1960) Cerebro-vascular disease. Part 5 (A comparative study of an American and a Norwegian population), World Neurology, 1 : 127. Baker, A. B., A. Iannone and J. Kinnard, (1961) Cerebro-vascular disease, Part 6 (Relationship to disease of the heart and the aorta), Neurology (Minneap.), 11: 63. Baker, A. B., S. Refsum and E. Dahl, (1960) Cerebro-vascular disease, Part 4 (A study of a Norwegian population). Neurology (Minneap.), 10: 525. DaMbska. M., (1963) Altération artériosclérotique cérébrale et artériosclérose des artères basales du cerveau. Acta neuropath. (Beri.), 2: 407. Kikk, J. E., (1951) Metabolism of arterial tissue (In Proceedings of the Symposium: Studies on A therosclerosis), J. Geront., 6: 167. Kraśnicka, Z., (1963) On the clinico-pathological correlation in various states of cerebral athero­ matosis (in Polish), Neuropat. pol., in the press. J. neuro!. Sci. (1964) 1: 13-23 http://rcin.org.pl ATHEROMA OF THE LARGER ARTERIES OF THE BRAIN IN A POLISH POPULATION 23 Majdecki, T., (1963) Atheromatosis of the large arteries of the brain basis and atheromatous changes on aorta and coronary arteries (in Polish), Neuropat. pol., in the press. Majdecki, T. and I. Zelman, (1961) Atheromatosis of the large arteries of the brain basis and atheromatous changes on aorta and coronary arteries, Part 1 (in Polish), Pol. med. Weekly, 16:1151. Mandybur, T., (1963) The atheromatous changes on the intracerebral arteries, Neuropat. pol., in the press. Ramsey, E. M., (1936/37) Studies on the pathology of vascular disease, nutrition of the blood vessel wall, Yale, J. Biol. Med., 9: 14, 36. Wiśniewski, W., A comparative study of the cerebral and renal arteries in cases of atheromatosis with and without hypertension, with respect to parenchymatous changes in the kidney. Neuro­ pat. pol., in the press. World Federation of Neurology, (1959) A Collaborative Study of Epidemiological FactorsinCerebro-Vascular Disease. Coding Guide, Antwerp. J. neurol. Sci. (1964) 1: 13-23 http://rcin.org.pl 

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MAJOR ARTICLE José Afonso Guerra-Assunção,1 Rein M. G. J. Houben,1 Amelia C. Crampin,1,2 Themba Mzembe,2 Kim Mallard,3 Francesc Coll,3 Palwasha Khan,1 Louis Banda,2 Arthur Chiwaya,2 Rui P. A. Pereira,3 Ruth McNerney,3 David Harris,4 Julian Parkhill,4 Taane G. Clark,1,3 and Judith R. Glynn1 1 Faculty of Epidemiology and Population Health, London School of Hygiene and Tropical Medicine, 2Karonga Prevention Study, Malawi, 3Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, and 4Wellcome Trust Sanger Institute, Hinxton, United Kingdom Background. Recurrent tuberculosis is a major health burden and may be due to relapse with the original strain or reinfection with a new strain. Methods. In a population-based study in northern Malawi, patients with tuberculosis diagnosed from 1996 to 2010 were actively followed after the end of treatment. Whole-genome sequencing with approximately 100-fold coverage was performed on all available cultures. Results of IS6110 restriction fragment-length polymorphism analyses were available for cultures performed up to 2008. Results. Based on our data, a difference of ≤10 single-nucleotide polymorphisms (SNPs) was used to define relapse, and a difference of >100 SNPs was used to define reinfection. There was no evidence of mixed infections among those classified as reinfections. Of 1471 patients, 139 had laboratory-confirmed recurrences: 55 had relapse, and 20 had reinfection; for 64 type of recurrence was unclassified. Almost all relapses occurred in the first 2 years. Human immunodeficiency virus infection was associated with reinfection but not relapse. Relapses were associated with isoniazid resistance, treatment before 2007, and lineage-3 strains. We identified several gene variants associated with relapse. Lineage-2 (Beijing) was overrepresented and lineage-1 underrepresented among the reinfecting strains (P = .004). Conclusions. While some of the factors determining recurrence depend on the patient and their treatment, differences in the Mycobacterium tuberculosis genome appear to have a role in both relapse and reinfection. Keywords. tuberculosis; relapse; reinfection; HIV; whole-genome sequence; RFLP; recurrence. Recurrent tuberculosis adds considerably to the burden of tuberculosis [1]. It arises through relapse of the original infection or through reinfection with a new strain. Received 21 July 2014; accepted 4 October 2014; electronically published 21 October 2014. Correspondence: Judith R. Glynn, PhD, FRCP, Faculty of Epidemiology and Population Health, London School of Hygiene and Tropical Medicine, London, UK (judith.glynn@lshtm.ac.uk). The Journal of Infectious Diseases® 2015;211:1154–63 © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Societyof America. 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. DOI: 10.1093/infdis/jiu574 1154 • JID 2015:211 (1 April) • Guerra-Assunção et al The 2 mechanisms have different implications for the patient and the control program. High rates of relapse may indicate unsuccessful treatment (relapse usually occurs early after the end of treatment and is associated with cavitary disease and drug resistance) [2–4]. A high rate of recurrence due to reinfection suggests a failure to develop protective immunity after the first episode and is associated with human immunodeficiency virus (HIV) infection [2, 3, 5]. Distinguishing the 2 mechanisms requires molecular marker analysis of cultures from both episodes, so results are available from only a few studies. These have shown varying proportions of disease due to reinfection [6–8], from <3% [9] to >60% [10, 11]. High proportions Downloaded from https://academic.oup.com/jid/article/211/7/1154/2910506 by guest on 21 May 2024 Recurrence due to Relapse or Reinfection With Mycobacterium tuberculosis: A Whole-Genome Sequencing Approach in a Large, PopulationBased Cohort With a High HIV Infection Prevalence and Active Follow-up METHODS Setting and Patients The Karonga Prevention Study has been studying tuberculosis in the whole of Karonga district, northern Malawi, since 1986 [14, 15]. The area is largely rural, with a population of around 300 000 and a prevalence of HIV infection among adults of about 10%. Individuals with tuberculosis are identified through enhanced passive surveillance, with project staff based at health centers to identify those with chronic cough. Sputum and other specimens (eg, lymph node aspirates) are processed in the project laboratory. Cultures that appear to be positive for M. tuberculosis are sent to the United Kingdom for species confirmation and drug resistance testing, initially for resistance to isoniazid and rifampicin and, if resistant to these, then for resistance to further drugs. At least 3 sputum samples are obtained at the time of diagnosis, with further samples collected at 2 months and at the end of treatment. Ethics approval for the study was given by the ethics committee of the London School of Hygiene and Tropical Medicine and the Malawian National Health Science Research Committee. Informed consent was sought from each participant. The incidence of smear-positive tuberculosis in adults in this district peaked at 125 cases/100 000 in the mid 1990s and then fell to 80–90 cases/100 000 [15]. The annual risk of infection with M. tuberculosis in the 1980s and 1990s was estimated to be 1% per year [14]. Since 1988, HIV testing has been offered to all patients with tuberculosis, following counseling and consent; nearly two thirds are HIV positive. Free antiretroviral therapy (ART) has been available in the district since 2005 to individuals with World Health Organization stage 3 or 4 disease (which includes tuberculosis) or with CD4+ T-cell counts of <250 cells/mL. Patients are treated according to Malawi government guidelines. Beginning in 1996, new smear-positive patients received a 2-month course of treatment with streptomycin, isoniazid, rifampicin, and pyrazinamide (2SHRZ) and a 6-month regimen of ethambutol and isoniazid (6HE) therapy. Ethambutol replaced streptomycin beginning in 2001. New smear-negative patients were treated with 1SEH/11HE until 2001, after which treatment was the same as that for smear-positive patients. Since January 2007, a 6-month regimen involving rifampicin throughout has been used for all new patients (2HRZE/4HR). Beginning in 1996, individuals requiring retreatment received a regimen containing rifampicin throughout [15]. Individuals were defined as having laboratory-confirmed tuberculosis if they had positive smears (excluding those with single scanty smears) or M. tuberculosis–positive cultures or histological evidence of tuberculosis. Outcomes beyond the end of treatment were ascertained by linkage to our other studies in the district and by home visits in 2005 to those whose vital status was unknown [16]. Since 2007 all patients have been visited annually, with sputum specimens collected from those who are symptomatic. Recurrences were defined as further episodes of diagnosed tuberculosis after the end of treatment, whether or not there was microbiological evidence of cure of the previous episode. Recurrences could be laboratory confirmed or not. Molecular Methods From 1996 to 2008, IS6110 RFLP analysis was done on all cultures [17, 18]. Whole-genome sequencing was carried out on all available cultures from 1996 to 2010 (n = 1933) at the Sanger Institute, using the Illumina HiSeq 2000 system, with paired-end reads of 100 base pairs. For each of the samples, Trimmomatic software (http://www.usadellab.org/cms/?page=trimmomatic) was used to remove low-quality sequences and low-quality 3′ ends of reads, retaining only reads at least 50 base pairs long, with nucleotides above quality score Q27 (equivalent to a risk of error of <0.2% per read per base pair). The trimmed sequence reads for each sample were mapped against the H37Rv reference genome (GenBank accession no. AL123456.3) and other publicly available mycobacterial genomes, using the BWA-mem algorithm (http://bio-bwa.sourceforge.net/). Samples (n = 79) Infection Relapse or Reinfection With M. tuberculosis • JID 2015:211 (1 April) • 1155 Downloaded from https://academic.oup.com/jid/article/211/7/1154/2910506 by guest on 21 May 2024 of apparent reinfections may be due to mislabeling, to crosscontamination or mixed infections, or to differences in patient populations, length of follow-up [7], and background risk of infection with Mycobacterium tuberculosis. A high proportion of apparent relapses could indicate insufficient variability of strains in the population with the marker used. Most studies to date have used IS6110-based restriction fragment-length polymorphism (RFLP) analysis or mycobacterial interspersed repetitive unit–variable number of tandem repeat (MIRUVNTR) typing [12]. Whole-genome sequencing gives greater discrimination than RFLP analysis or MIRU-VNTR analysis, which should improve the ability to distinguish relapse from reinfection. The one study to date that evaluated whole-genome sequencing for this purpose compared whole-genome sequencing data to MIRUVNTR data, resulting in reclassification of several strains and allowing detection of mixed infections [12]. Whole-genome sequencing also allows genetic determinants of recurrence to be examined. In a long-term population-based study of tuberculosis in northern Malawi with active follow-up beyond the end of treatment, we previously analyzed RFLP data from 1996 to 2005 and found that HIV infection increased the risk of recurrence due to reinfection but not the risk due to relapse [2]. Here, we have now used whole-genome sequences of all available strains obtained from patients up to 2010, and assessed the effect of HIV, M. tuberculosis lineage [13], and other factors on the rate of recurrence due to reinfection or relapse. Mixed Infections To explore whether any of the apparent reinfections were explained by the presence of a mixed infection, the number of heterozygous positions was calculated. A position was classified as heterozygous if >1 allele accounted for ≥30% of the reads (and there were >30 reads). for recurrence was censored at the date of death, leaving the district, or the first recurrence of tuberculosis, whether confirmed or not (since those receiving treatment are not at risk). HIV status was treated as a time-varying covariate for those who were HIV negative or had an unknown HIV status during the first episode. The date of seroconversion was taken as halfway between the last negative test result (or the end of the first episode if the HIV status during that episode was unknown) and the first positive test result (or the date of ART initiation if this was the first evidence of HIV positivity). Calendar time and age were also treated as time-varying covariates. Survival methods and Nelson-Aalen plots of cumulative rates were used. Rate ratios were calculated using Poisson regression, stratifying by time since the end of treatment. STATA 13 was used for all statistical analyses. Risk factors for relapse and reinfection were examined separately. RESULTS Differentiating Relapse and Reinfection After quality control, 1718 whole-genome sequences were available for analysis. These included some individuals with >1 sample from the same or different disease episode. For each of these individuals, the first and last sample were selected if there were >2 samples available. This left 92 individuals with paired samples, including 66 with samples from different episodes of disease. The number of SNPs between each of the pairs is shown in Figure 1. There is a clear gap, with pairs having a difference of ≤8 SNPs or >100 SNPs. RFLPs were available for both members of 42 pairs. All pairs (7 of 7) with a difference of >100 SNPs had different RFLPs. All pairs (35 of 35) with ≤8 SNPs different had identical RFLPs (n = 28) or RFLPs that differed by only 1 band (n = 7). Genomic Associations To explore genomic associations with relapse, the genomes from the index episode of all individuals with molecularly confirmed relapses were compared with those from patients who were actively followed up and had no evidence of recurrence. Our in-house implementation of the PhyC algorithm [22] was used to explore associations showing evidence of positive selection, using a phylogenetic tree produced by RAxML [23]. Genome-wide association analysis for SNPs and annotated protein coding genes was performed using R statistical software, using logistic regression and including lineage as a covariate. For each gene, the number of mutations was aggregated by individual. A Bonferroni cutoff based on the number of independent genes with SNPs (3689) suggested a multiple testing– adjusted significance threshold of 0.000014. Epidemiological Analysis Rates of laboratory-confirmed recurrence were calculated from the end of treatment to the end of June 2011. The time at risk 1156 • JID 2015:211 (1 April) • Guerra-Assunção et al Figure 1. Number of single-nucleotide polymorphisms between pairs of samples from individuals from the same episode of tuberculosis or from an index episode and a recurrent episode. Downloaded from https://academic.oup.com/jid/article/211/7/1154/2910506 by guest on 21 May 2024 were excluded if the best match was a non-tuberculous mycobacterium or if their average genomic coverage was <10-fold. The average coverage of the remaining samples was high (median, 88-fold [range, 12–608-fold]; mean, 127-fold). Single-nucleotide polymorphism (SNP) positions were identified using SAMtools (http://samtools.sourceforge.net/). Sample genotypes were called using the majority allele (minimum frequency, 75%) in positions supported by at least 20-fold total coverage; they were otherwise classified as missing. This strategy ignores heterozygous calls. Samples with >15% missing genotype calls were excluded, thereby removing those that may be contaminated, those containing mixed isolates, or those involving technical errors. Similarly, we excluded SNPs with >15% missing genotypes and those in highly repetitive and variable regions (eg, PE/PPE genes). In the final analysis, 94% of the M. tuberculosis genome was analyzed for variants. The final data set consists of 1718 samples (89% of the original cultures) and 40 046 SNPs. Raw sequence data are available from the European Nucleotide Archive (accession numbers ERP000436 and ERP001072). Spoligotyping was performed in silico using SpolPred [19]. Lineages were defined from spoligotype families [20]. For samples with RFLP results but no whole-genome sequence data available, lineage was deduced from spoligotypes of these specimens or others with the same RFLP within the data set, as previously described [21]. Selection of patients. Abbreviations: RFLP, restriction fragment-length polymorphism data; WGS, whole-genome sequence data. For the main analysis, only pairs from different episodes were used. Recurrence was defined as being due to relapse if there was a difference of ≤10 SNPs and as due to reinfection if there was a difference of >100 SNPs. For individuals with recurrent episodes with RFLP but no whole-genome sequencing data available, the RFLP data were used to define relapse and reinfection. Relapses had identical patterns or differed by 1 band. Reinfections had completely different patterns. In the whole data set, the number of heterozygous positions was low for most samples, with a few having large numbers of heterozygotes. From inspection of the plot of the number of heterozygotes per sample (Supplementary Figure 1), a cutoff of 140, the inflection point in the empirical distribution of the data, was chosen to identify possible mixtures. None of the reinfections had evidence of mixed infections in either episode of disease. Two of the patients with subsequent relapse had evidence of a mixed infection in the first episode (679 and 945 heterozygous positions). All further analysis was restricted to individuals who received a diagnosis of laboratory-confirmed tuberculosis between January 1996 and February 2010, who were normally resident in Karonga district, and who had completed treatment. There were 1535 such individuals (Figure 2). Information beyond the end of treatment was available for 1471 (96%). Of these, 139 had laboratory-confirmed recurrences, and 64 had unconfirmed recurrences. Of the confirmed recurrences, 55 were due to relapse (defined on the basis of SNP data in 46 cases and RFLP data in 9 cases), and 20 were due to reinfection (defined on the basis of SNP data in 14 cases and RFLP data in 6 cases); 64 were unclassified (no paired samples were available). The 64 Infection Relapse or Reinfection With M. tuberculosis • JID 2015:211 (1 April) • 1157 Downloaded from https://academic.oup.com/jid/article/211/7/1154/2910506 by guest on 21 May 2024 Figure 2. Table 1. Rate of Tuberculosis Recurrence, by Patient and Index Tuberculosis Episode Characteristics Rate of Recurrence per 100 Person-Years at Risk During the First 2 Years All Recurrences (Relapse, Reinfection) Person-Years at Risk Overall 98 (51, 8) Age, y <30 Characteristic After the First 2 Years Rate (95% CI) All Recurrences (Relapse, Reinfection) Person-Years at Risk Rate (95% CI) 2311 4.2 (3.5–5.2) 41 (4, 12) 3995 1.0 (.76–1.4) 0.92 (.44–1.9) 671 3.3 (2.2–5.0) 7 (2, 0) 758 35 (17, 2) 770 4.5 (3.3–6.3) 16 (0, 7) 1314 1.2 (.75–2.0) 40–49 ≥50 19 (9, 0) 22 (12, 2) 417 452 4.6 (2.9–7.1) 4.9 (3.2–7.4) 7 (1, 1) 11 (1, 4) 803 1051 0.80 (.38–1.7) 1.0 (.58–1.9) 43 (25, 6) 55 (26, 2) 1246 1065 3.5 (2.6–4.7) 5.2 (4.0–6.7) 28 (1, 7) 18 (3, 5) 2116 1879 1.1 (.72–1.6) 0.96 (.60–1.5) Negative Positive 35 (21, 0) 59 (29, 7) 922 1087 3.8 (2.7–5.3) 5.4 (4.2–7.0) 9 (1, 1) 31 (3, 11) 1978 1412 0.46 (.24–.87) 2.2 (1.5–3.1) Unknown 4 (1, 1) 302 1.3 (.50–3.5) 1 (0, 0) 605 0.17 (.02–1.2) 82 (47, 4) 1894 4.3 (3.5–5.4) 35 (3, 10) 3244 1.1 (.77–1.5) 13 (3, 3) 269 4.8 (2.8–8.3) 4 (1, 1) 440 0.91 (.34–2.4) 3 (1, 1) 148 2.0 (.65–6.3) 2 (0, 1) 310 0.65 (.16–2.6) 90 (49, 7) 2167 4.2 (3.4–5.1) 38 (4, 11) 3759 1.0 (.74–1.4) 8 (2, 1) 144 5.5 (2.8–11.1) 3 (0, 1) 235 1.3 (.41–4.0) Sex Female Male HIV status Tuberculosis type Smear-positive pulmonary Smear-negative pulmonary Extrapulmonary Previous tuberculosis No Yes M. tuberculosis lineage 1 11 (6, 1) 270 4.1 (2.3–7.4) 8 (0, 3) 468 1.7 (.85–3.4) 2 3 1 (1, 0) 22 (16, 0) 70 167 1.4 (.20–10.1) 13.2 (8.7–20.0) 1 (0, 1) 1 (0, 0) 156 212 0.64 (.09–4.5) 0.47 (.07–3.4) 4 41 (27, 6) 1192 3.4 (2.5–4.7) 25 (4, 8) 2158 1.2 (.78–1.7) 23 (1, 1) 614 3.7 (2.5–5.6) 6 (0, 0) 1000 0.60 (.27–1.3) No 76 (44, 8) 1843 4.1 (3.3–5.2) 33 (3, 9) 3344 1.0 (.70–1.4) Yes Unknown 9 (6, 0) 13 (1, 0) 81 387 11.1 (5.8–21.4) 3.4 (2.0–5.8) 4 (1, 3) 4 (0, 0) 109 542 3.7 (1.4–9.8) 0.74 (.28–2.0) 1996–2000 2001–2004 25 (11, 6) 26 (19, 2) 589 629 4.2 (2.9–6.3) 4.1 (2.8–6.1) 3 (0, 1) 11 (2, 4) 151 1042 2.0 (.64–6.2) 1.1 (.58–1.9) 2005 and later 47 (21, 0) 1093 4.3 (3.2–5.7) 27 (2, 7) 2802 0.96 (.66–1.4) 80 (47, 8) 1786 4.5 (3.6–5.6) 39 (4, 12) 3862 1.0 (.74–1.4) 18 (4, 0) 525 3.4 (2.2–5.4) 2 (0, 0) 132 1.5 (.38–6.0) Unknown Isoniazid resistance Year of follow-up Year of treatment initiation Before 2007 2007 and later Abbreviations: CI, confidence interval; HIV, human immunodeficiency virus; M. tuberculosis, Mycobacterium tuberculosis. unclassified recurrences were similar to those that could be classified in terms of age, sex, HIV status, and year. Recurrence The rate of recurrence decreased by time since the end of the index episode. There were 71 recurrences in the first year, for a rate of 5.4 recurrences/100 person-years at risk (95% 1158 • JID 2015:211 (1 April) • Guerra-Assunção et al confidence interval [CI], 4.3–6.8); 27 in the second year, for a rate of 2.7/100 person-years at risk (95% CI, 1.8–3.9); and 41 after the second year, for a rate of 1.0/100 person-years at risk (95% CI, .76–1.4). The factors associated with recurrence within the first 2 years and beyond are shown in Table 1, and the development of recurrences, relapses, and reinfections over time are shown in Figure 3. Almost all known relapses (51 of 55) Downloaded from https://academic.oup.com/jid/article/211/7/1154/2910506 by guest on 21 May 2024 22 (13, 4) 30–39 occurred in the first 2 years, whereas reinfections occurred at a more constant rate over time. Relapse Because of the very low number and rate of (confirmed) relapses after 2 years, risk factors for relapse were investigated within the first 2 years (Table 2). Relapses were more common soon after the end of treatment. They were not associated with age, sex, or HIV status. Fifty-five patients had strains that were isoniazid resistant in the index episode (including 5 that were also rifampicin resistant). Patients with isoniazid-resistant strains had 3 times the rate of relapse of those who had isoniazid-susceptible strains. Those who started treatment after 1 January 2007, who would have received rifampicin throughout their treatment, had much lower rates of relapse (rate ratio [RR], 0.29 [95% CI, .10–.80]). Those with lineage-3 strains had much higher relapse rates than those with strains from other lineages (RR, 4.2 [95% CI, 2.3–7.8], compared with lineage-4). All of these associations persisted after adjustment for each other, and additional adjustment for the other factors made no difference to the results (Table 2). The genomes of isolates from the index tuberculosis episodes of 52 patients with relapse were compared with those from 843 patients with active follow-up and no recurrence of tuberculosis. An analysis of SNPs showing evidence of convergent evolution and positive selection based on the PhyC algorithm found 9 SNPs (from 8 genes) that were significant at the 5% level (Supplementary Table 1). Six SNPs overlapped protein-coding genes and caused nonsynonymous changes. These include the known isoniazid-resistance associated gene, katG. Genome-wide association analyses showed no significant genic results, after correction for multiple testing. Supplementary Table 2 lists all genes identified at a less stringent threshold (P < .05); this includes katG. Reinfection The main factor associated with reinfection was HIV status (Table 2). Among HIV-negative individuals, only 1 of 44 recurrences was due to reinfection, 22 were relapses, and 21 could not be classified. Among HIV-positive individuals, 18 of 90 recurrences were due to reinfection, 32 were relapses, and 40 could Infection Relapse or Reinfection With M. tuberculosis • JID 2015:211 (1 April) • 1159 Downloaded from https://academic.oup.com/jid/article/211/7/1154/2910506 by guest on 21 May 2024 Figure 3. Cumulative hazard of confirmed recurrent tuberculosis overall (A) and by human immunodeficiency virus (HIV) status (B ). C and D, Cumulative hazard for recurrences due to relapse and reinfection, by HIV status. Table 2. Relative Rates for Relapse (Within 2 Years) and Reinfection (At Any Time) Rate Ratio for Reinfection Disease (95% CI) Rate Ratio for Relapse Within 2 Years (95% CI) Univariable Multivariablea <30 30–39 1 1.1 (.55–2.3) 40–49 Characteristic P Valueb Univariable Multivariablea ... ... 1 1.5 (.48–5.0) ... ... 1.1 (.48–2.6) ... 0.28 (.03–2.5) ... 1.4 (.63–3.0) ... 1.4 (.40–5.1) ... P Valueb Age, y, at start ≥50 Sex Female 1 Male HIV status ... 1.2 (.70–2.1) 1 Positive Unknown ... 0.61 (.25–1.5) ... 1 ... <.001c 1 1.2 (.67–2.1) 0.15 (.02–1.1) ... ... 20.9 (2.8–156) 3.2 (.20–51.1) 21.0 (2.8–157) 2.5 (.15–40.2) Smear-positive pulmonary Smear-negative pulmonary 1 0.45 (.14–1.4) ... ... 1 2.1 (.68–6.3) ... ... Extrapulmonary 0.27 (.04–2.0) ... 1.6 (.36–6.3) ... Tuberculosis type Previous tuberculosis No 1 Yes ... 0.61 (.15–2.5) ... 0.99 (.41–2.4) 1.0 (.42–2.4) 2 0.63 (.09–4.6) 0.65 (.09–4.8) 3 4 4.2 (2.3–7.8) 1 4.2 (2.2–7.8) 1 0.07 (.01–.53) 0.09 (.009–.87) M. tuberculosis lineage 1 Unknown Isoniazid resistance No 1 Yes Unknown Calendar year 1996–2000 .004c ... ... 1.3 (.43–3.9) ... 1.1 (.14–8.0) ... ... ... ... 0.15 (.02–1.1) ... 1 .05c 1 1 .06c 1 3.1 (1.3–7.3) 2.6 (1.1–6.0) 4.8 (1.4–16.5) 4.1 (1.2–14.0) 0.11 (.01–.79) 0.91 (.09–9.1) ... ... 0.38 (.13–1.1) 0.19 (.07–0.54) 0.20 (.06–.66) 0.10 (.03–.32) 1 2001–2004 2005 and later 1 1.7 (.40–7.5) ... 1.6 (.77–3.4) 1.0 (.50–2.1) ... ... 1 0.40 (.21–.77) 1 0.39 (.20–.74) ... ... 0.29 (.10–.80) 0.29 (.10–.83) 1 1 .001 Follow-up First year Second year Third year and later Year of treatment initiation Before 2007 1 2007 and later ... .002 .006 1 3.9 (.79–19.4) 1 4.7 (.94–23.3) 2.0 (.44–8.8) 5.9 (1.2–29.7) ... ... ... ... .03 Abbreviations: CI, confidence interval; HIV, human immunodeficiency virus; M. tuberculosis, Mycobacterium tuberculosis. a Adjusted for the other factors shown in the multivariable model. b From the likelihood ratio χ2 test. c From the model excluding unknowns for this variable. not be unclassified. The one HIV-negative patient with reinfection was confirmed as being HIV negative at the time of the second episode. Reinfections were less common in more recent calendar years and were less common in the first year after 1160 • JID 2015:211 (1 April) • Guerra-Assunção et al the end of treatment than they were subsequently. These associations became slightly stronger after adjustment for each other, with no further change after adjustment for the other available factors. Isoniazid resistance in the first episode was Downloaded from https://academic.oup.com/jid/article/211/7/1154/2910506 by guest on 21 May 2024 Negative 1 ... DISCUSSION Whole-genome sequencing of strains from initial and recurrent episodes of disease showed a clear distinction between those with few (0–8) different SNPs and those with many (>100) differences. That this was an appropriate cutoff for distinguishing relapse and reinfection is supported by the distribution of SNP differences within the same episode (Figure 1), by the RFLP results, and by the strong correlation of relapse and reinfection so defined with time since the end of treatment and with HIV status. The cutoff is slightly higher than suggested in some other studies of SNP differences within the same episode or during transmission between patients [24–26], but there is considerable uncertainty about the appropriate cutoff, and the value will vary depending on the pipeline used and the genome coverage achieved [26]. The correlation between SNP and RFLP results suggests that an RFLP that is identical or differs by 1 band is a good approximation for defining relapse when no sequencing data are available. In this study, none of the reinfections were explained by mixed infection. This is in contrast to findings from Cape Town, where the annual risk of infection with M. tuberculosis is much higher [12, 27]. We have previously found a low proportion of mixed infections in this population [28]. The timing and risk factors for relapse and reinfection in this population are strikingly different. Relapse was most common in the first year and rare after the second year. Recurrences due to reinfection were less common in the first year, but then the rate was approximately constant. HIV infection greatly increased the rate of recurrence due to reinfection but was not associated with relapse. Calendar year was also associated with recurrence due to reinfection, with higher rates before 2001, when the incidence of new tuberculosis cases was also higher [15]. Relapse was associated with isoniazid resistance (as expected [29]), treatment in earlier years (before rifampicin was given in the continuation phase, consistent with the reported effect of prolonged rifampicin on recurrence [30]), and lineage. There is increasing interest in the extent to which genomic variation in M. tuberculosis may explain the variations seen in disease occurrence, patterns, and outcomes. Several studies have shown an increased rate of relapse for lineage-2 (Beijing) strains [31–33]. We did not find this, but there were only 43 patients with lineage-2 strains in the index episode, so study power was limited. We found greatly increased relapse rates for those with lineage-3 strains (East African Indian/CAS), independent of other factors. This has not been described before. In a recent study from Vietnam [33], relapse rates were compared between the Beijing strain and all other strains; lineage-3 made up about half of the comparison group. Even if all of the relapses had occurred in patients with this lineage, the rate would have been lower than the rate for those with Beijing strains. Using the PhyC algorithm and genome-wide association analysis, we found no genomic variants strongly associated with relapse. The expected finding of associations with katG, which is known to be involved in isoniazid resistance, supports the validity of the methods and suggests that the other identified genes should be investigated further for clues for mechanisms for relapse. Although this is one of the largest studies to compare paired samples to differentiate relapse and reinfection, power is limited for genetic comparisons of this type. The lack of paired samples for 46% of the recurrences (64 of 139) will have led to underestimation of relapse and reinfection rates but is unlikely to have biased the results of associations with relapse and reinfection, as individuals without paired samples were similar to those with paired samples. In the only previous study to use whole-genome sequencing to look at reinfection, it was suggested, based on 3 reinfections, that reinfections with strains differing by >1000 SNPs (representing different lineages) might be more likely [12]. In this analysis, 9 of 19 patients with reinfections with the lineage known for both episodes had reinfections with the same lineage, which does not support this hypothesis. However, there was some evidence that the lineage of the reinfecting strain might be important. Among episodes of recurrence due to reinfection, we found that lineage-2 strains were overrepresented and that lineage-1 (Indo-Oceanic) strains were underrepresented. This strengthens our earlier observations in this population [2] and is consistent with suggested higher virulence for lineage-2 strains [34] and lower virulence of lineage-1 strains [21]. Other studies of reinfection have not reported the results by lineage. The use of whole-genome sequencing surmounts some of the potential problems with older techniques, namely, excluding mixed infections and knowing how much change to allow Infection Relapse or Reinfection With M. tuberculosis • JID 2015:211 (1 April) • 1161 Downloaded from https://academic.oup.com/jid/article/211/7/1154/2910506 by guest on 21 May 2024 also weakly associated with recurrence due to reinfection (Table 2). Of the 20 strains causing reinfection, 1 (5%) was lineage-1, 5 (25%) were lineage-2, 2 (10%) were lineage-3, and 12 (60%) were lineage-4. This compares with a distribution of lineages of 16%, 4%, 11%, and 68%, respectively, for the 4 lineages for the index episode (P = .004, by the Fisher exact test). The proportion of index episodes due to lineages 1 and 2 changed little over the study, so this did not explain the difference. Of the 19 patients with reinfection for whom the lineage was known in both episodes, 10 (53%) had different lineages in the 2 episodes (compared with 48% expected by chance, given the lineage distribution in the population). For the 14 patients with reinfection defined on the basis of SNP data, the number of differing SNPs was 100–200 for 4 patients, 600–800 for 2 patients, and >1000 for 8 patients. All those with <1000 differing SNPs had lineage4 strains in both episodes. All those with a difference of >1000 SNPs had different lineages in the 2 episodes. The one reinfection in an HIV-negative patient was with a lineage-2 strain, following lineage-1 in the initial episode. when comparing specimens from the different episodes of disease. It validated our earlier results with RFLP analysis [2] and allows the genomic associations with relapse and reinfection to be examined directly. In this large population-based study with active follow-up, relapses were rare after the first 2 years and were associated with drug resistance and lineage-3 genotypes. Reinfections occurred at a more constant rate, were largely restricted to HIV-positive individuals, and were not more likely to be due to different lineages from the index episode but were due to lineage-2 genotypes more often than would be expected by chance. Supplementary Data Notes Acknowledgments. We thank the Government of the Republic of Malawi, for their interest in this project, and the National Health Sciences Research Committee of Malawi, for permission to submit the manuscript for publication. Financial support. This work was supported by the Wellcome Trust (grant 096249/Z/11/B). Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. 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Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author. 10. Shen G, Xue Z, Shen X, et al. Recurrent tuberculosis and exogenous reinfection, Shanghai, China. Emerg Infect Dis 2006; 12:1776–8. 11. van Rie A, Warren R, Richardson M, et al. Exogenous reinfection as a cause of recurrent tuberculosis after curative treatment. N Engl J Med 1999; 341:1174–9. 12. Bryant JM, Harris SR, Parkhill J, et al. Whole-genome sequencing to establish relapse or re-infection with Mycobacterium tuberculosis: a retrospective observational study. Lancet Respir Med 2013; 1:786–92. 13. Comas I, Homolka S, Niemann S, Gagneux S. Genotyping of genetically monomorphic bacteria: DNA sequencing in mycobacterium tuberculosis highlights the limitations of current methodologies. PLoS One 2009; 4:e7815. 14. Crampin AC, Glynn JR, Fine PE. What has Karonga taught us? Tuberculosis studied over three decades. Int J Tuberc Lung Dis 2009; 13:153–64. 15. Mboma SM, Houben RM, Glynn JR, et al. Control of (multi)drug resistance and tuberculosis incidence over 23 years in the context of a wellsupported tuberculosis programme in rural Malawi. PLoS One 2013; 8: e58192. 16. Houben RM, Glynn JR, Mboma S, et al. The impact of HIV and ART on recurrent tuberculosis in a sub-Saharan setting. AIDS 2012; 26:2233–9. 17. Glynn JR, Crampin AC, Yates MD, et al. The importance of recent infection with M tuberculosis in an area with high HIV prevalence: a longterm molecular epidemiological study in northern Malawi. J Infect Dis 2005; 192:480–7. 18. Glynn JR, Yates MD, Crampin AC, et al. DNA fingerprint changes in tuberculosis: re-infection, evolution, or laboratory error? J Infect Dis 2004; 190:1158–66. 19. Coll F, Mallard K, Preston MD, et al. SpolPred: rapid and accurate prediction of Mycobacterium tuberculosis spoligotypes from short genomic sequences. Bioinformatics 2012; 28:2991–3. 20. Demay C, Liens B, Burguiere T, et al. SITVITWEB–a publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology. Infect Genet Evol 2012; 12:755–66. 21. Glynn JR, Alghamdi S, Mallard K, et al. Changes in Mycobacterium tuberculosis genotype families over 20 years in a population-based study in Northern Malawi. PLoS One 2010; 5:e12259. 22. Farhat MR, Shapiro BJ, Kieser KJ, et al. Genomic analysis identifies targets of convergent positive selection in drug-resistant Mycobacterium tuberculosis. Nat Genet 2013; 45:1183–9. 23. Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 2014; 10.1093/bioinformatics/btu033. 24. Walker TM, Ip CL, Harrell RH, et al. Whole-genome sequencing to delineate Mycobacterium tuberculosis outbreaks: a retrospective observational study. Lancet Infect Dis 2013; 13:137–46. 25. Bryant JM, Schurch AC, van Deutekom H, et al. Inferring patient to patient transmission of Mycobacterium tuberculosis from whole genome sequencing data. BMC Infect Dis 2013; 13:110. 26. Perez-Lago L, Comas I, Navarro Y, et al. Whole genome sequencing analysis of intrapatient microevolution in Mycobacterium tuberculosis: potential impact on the inference of tuberculosis transmission. J Infect Dis 2014; 209:98–108. 27. van Rie A, Victor TC, Richardson M, et al. Reinfection and mixed infection cause changing Mycobacterium tuberculosis drug-resistance patterns. Am J Respir Crit Care Med 2005; 172:636–42. 28. Mallard K, McNerney R, Crampin AC, et al. Molecular detection of mixed infections of Mycobacterium tuberculosis in sputum samples from patients in Karonga District, Malawi. J Clin Microbiol 2010; 48:4512–8. 29. Mitchison DA, Nunn AJ. Influence of initial drug resistance on the response to short- course chemotherapy of pulmonary tuberculosis. Am Rev Respir Dis 1986; 133:423–30. 30. Korenromp EL, Scano F, Williams BG, Dye C, Nunn P. Effects of human immunodeficiency virus infection on recurrence of tuberculosis after rifampin-based treatment: an analytical review. Clin Infect Dis 2003; 37:101–12. 31. Lan NT, Lien HT, Tung le B, Borgdorff MW, Kremer K, van Soolingen D. Mycobacterium tuberculosis Beijing genotype and risk for treatment failure and relapse, Vietnam. Emerg Infect Dis 2003; 9:1633–5. 32. Burman WJ, Bliven EE, Cowan L, et al. Relapse associated with active disease caused by Beijing strain of Mycobacterium tuberculosis. Emerg Infect Dis 2009; 15:1061–7. 33. Huyen MN, Buu TN, Tiemersma E, et al. 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Downloaded from https://academic.oup.com/jid/article/211/7/1154/2910506 by guest on 21 May 2024 Infection Relapse or Reinfection With M. tuberculosis • JID 2015:211 (1 April) • 1163 EVIDENCE SUPPORTS THE HIGH BARRIER TO RESISTANCE OF DOVATO UP TO 5 YEARS1-3 >300,000 PEOPLE LIVING WITH HIV HAVE BEEN TREATED WITH DOVATO GLOBALLY10 CONFIDENCE IN DOVATO ACROSS TREATMENT SETTINGS4–9 Treatmentnaïve resistance rates, with up to years of evidence5–7 3 Treatmentexperienced resistance rates, with up to years of evidence1–3 5 0% (n=0/1,885)*,4 0.1% REALWORLD EVIDENCE RANDOMISED CONTROLLED TRIALS (n=1/953)**,†,‡,§,5–7 0.03% 0% (n=10/35,888) *,4 (n=0/615)||,¶,#,8,9 REALWORLD EVIDENCE RANDOMISED CONTROLLED TRIALS DOVATO is supported by a wealth of evidence, NO PRIOR TREATMENT EXPERIENCE13 with the outcomes of >40,000 people living with HIV captured within NO BASELINE RESISTANCE TESTING13 HIGH BASELINE VIRAL LOAD clinical trials and realworld evidence, including those with: 4–9,11,12 LOW CD4 + COUNT (>100,000 copies/mL and even >1M copies/mL) 6,13 (≤200 cells/mm 3) 13 Patients from phase III RCTs Patients from unique real-world cohorts >40,000 >34,000 >14,000 >100 2015 2016 >500 2017 >900 2018 >2,300 >4,100 2019 2020 >6,600 2021 2022 2023 2024 Year LEARN MORE DOVATO is indicated for the treatment of Human Immunodeficiency Virus type 1 (HIV-1) infection in adults and adolescents above 12 years of age weighing at least 40 kg, with no known or suspected resistance to the integrase inhibitor class, or lamivudine.13 Adverse events should be reported. Reporting forms and information can be found at https://yellowcard.mhra.gov.uk/ or search for MHRA Yellowcard in the Google Play or Apple App store. Adverse events should also be reported to GSK on 0800 221441 REFERENCES 1. 2. 3. 4. 5. 6. 7. Maggiolo F et al. BMC Infect Dis 2022; 22(1): 782. Taramasso L et al. AIDS Patient Care STDS 2021; 35(9): 342–353. Ciccullo A et al. JAIDS 2021; 88(3): 234–237. ViiV Healthcare. Data on File. REF-223795. 2024. Cahn P et al. AIDS 2022; 36(1): 39–48. Rolle C et al. Open Forum Infect Dis 2023; 10(3): ofad101. Cordova E et al. Poster presented at 12th IAS Conference on HIV Science. 23–26 July 2023. Brisbane, Australia. TUPEB02. 8. De Wit S et al. Slides presented at HIV Glasgow. 23–26 October 2022. Virtual and Glasgow, UK. M041. 9. Llibre J et al. Clin Infect Dis 2023; 76(4): 720–729. 10. ViiV Healthcare. Data on File. REF-220949. 2024. 11. Rolle C et al. Poster presented IDWeek. 11–15 October 2023. Virtual and Boston, USA. 1603. 12. Slim J et al. Abstract presented IDWeek. 11–15 October 2023. Virtual and Boston, USA. 1593. 13. DOVATO. Summary of Product Characteristics. June 2023. PRESCRIBING INFORMATION Dovato Prescribing Information Legal Notices Privacy Policy Contact Us ViiV Healthcare, 980 Great West Road, Brentford, Middlesex, London, UK. ViiV trademarks are owned by or licensed to the ViiV Healthcare group of companies. Non-ViiV trademarks are owned by or licensed to their respective owners or licensors. ©2024 ViiV Healthcare group of companies or its licensor. All rights reserved. Intended for healthcare professionals only. ABBREVIATIONS 3TC, lamivudine; CD4, cluster of differentiation 4; DTG, dolutegravir; FDA, United States Food and Drug Administration; FTC, emtricitabine; HIV, human immunodeficiency virus; ITT-E, intention-to-treat exposed; NRTI, nucleoside/nucleotide reverse transcriptase inhibitor; RCT, randomised controlled trial; RNA, ribonucleic acid; TAF, tenofovir alafenamide fumarate; TDF, tenofovir disoproxil fumarate; XTC, emtricitabine. FOOTNOTES *Data extracted from a systematic literature review of DTG+3TC real-world evidence. Overlap between cohorts cannot be fully excluded. **The reported rate reflects the sum-total of resistance cases calculated from GEMINI I and II (n=1/716, through 144 weeks), STAT (n=0/131, through 52 weeks), and D2ARLING (n=0/106, through 24 weeks).5–7 †GEMINI I and II are two identical 148-week, phase III, randomised, double-blind, multicentre, parallel-group, non-inferiority, controlled clinical trials testing the efficacy of DTG/3TC in treatment-naïve patients. Participants with screening HIV-1 RNA ≤500,000 copies/mL were randomised 1:1 to once-daily DTG/3TC (n=716, pooled) or DTG + TDF/FTC (n=717, pooled). The primary endpoint of each GEMINI study was the proportion of participants with plasma HIV-1 RNA <50 copies/mL at Week 48 (ITT-E population, snapshot algorithm).13 ‡STAT is a phase IIIb, open-label, 48-week, single-arm pilot study evaluating the feasibility, efficacy, and safety of DTG/3TC in 131 newly diagnosed HIV-1 infected adults as a first line regimen. The primary endpoint was the proportion of participants with plasma HIV-1 RNA <50 copies/mL at Week 24.6 §D2ARLING is a randomised, open-label, phase IV study designed to assess the efficacy and safety of DTG/3TC in treatment-naïve people with HIV with no available baseline HIV-1 resistance testing. Participants were randomised in a 1:1 ratio to receive DTG/3TC (n=106) or DTG + TDF/XTC (n=108). The primary endpoint was the proportion of participants with plasma HIV-1 RNA <50 copies/mL at Week 48.7 Results at week 24 of the study. ||The reported rate reflects the sum-total of resistance cases calculated from TANGO (n=0/369, through 196 weeks) and SALSA (n=0/246, through 48 weeks).8,9 ¶TANGO is a randomised, open-label, trial testing the efficacy of DOVATO in virologically suppressed patients. Participants were randomised in a 1:1 ratio to receive DOVATO (n=369) or continue with TAF-containing regimens (n=372) for up to 200 weeks. At Week 148, 298 of those on TAF-based regimens switched to DOVATO. The primary efficacy endpoint was the proportion of subjects with plasma HIV-1 RNA ≥50 copies/mL (virologic non-response) as per the FDA Snapshot category at Week 48 (adjusted for randomisation stratification factor).8,13 #SALSA is a phase III, randomised, open-label, non-inferiority clinical trial evaluating the efficacy and safety of switching to DTG/3TC compared with continuing current antiretroviral regimens in virologically suppressed adults with HIV. Eligible participants were randomised 1:1 to switch to once-daily DTG/3TC (n=246) or continue current antiretroviral regimens (n=247). The primary endpoint was the proportion of subjects with plasma HIV-1 RNA ≥50 copies/mL at Week 48 (ITT-E population, snapshot algorithm).9 PM-GBL-DLL-BNNR-240004 May 2024 

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International Journal of Education, Social Science & Humanities. FARS Publishers Impact factor (SJIF) = 6.786 Volume-11| Issue3| 2023 Research Article BO`LAJAK TARIX FANI O`QITUVCHILARINING KASBIY VA DIDAKTIK MADANIYATINI SHAKLLANTIRISH https://doi.org/10.5281/zenodo.7769221 G.B.Shertaylakova JizDPU, mustaqil taqiqotchi Received: 22-03-2023 Accepted: 22-03-2023 Published: 22-03-2023 Received: 22-03-2023 Accepted: 22-03-2023 Published: 22-03-2023 Received: 22-03-2023 Accepted: 22-03-2023 Published: 22-03-2023 Abstract: Ushbu maqolada bo`lajak tarix fani o`qituvchilarining kasbiy va didaktik madaniyatini shakllantirish muammolariga qaratilgan bo`lib, unda muammolar o`rganishda yetakchi yondashuvlar asosida yoritilgan. Keywords: didaktika, didaktik madaniyat, kasbiy madaniyat, didaktik faoliyat, tizimli-kulturologik yondashuv, subyekt komponenti. About: FARS Publishers has been established with the aim of spreading quality scientific information to the research community throughout the universe. Open Access process eliminates the barriers associated with the older publication models, thus matching up with the rapidity of the twenty-first century. Abstract: This article focuses on the problems of forming the professional and didactic culture of future teachers of history, in which problems are covered on the basis of leading approaches to learning. Keywords: didactic, didactic culture, professional culture, didactic activity, systematic-culturological approach, subject component. About: FARS Publishers has been established with the aim of spreading quality scientific information to the research community throughout the universe. Open Access process eliminates the barriers associated with the older publication models, thus matching up with the rapidity of the twenty-first century. Abstract: Данная статья посвящена проблемам формирования профессиональной и дидактической культуры будущих учителей истории, в которой проблемы освещаются на основе ведущих подходов к обучению. Keywords: дидактика, дидактическая культура, профессиональная культура, дидактическая деятельность, системно-культурологический подход, предметный компонент. About: FARS Publishers has been established with the aim of spreading quality scientific information to the research community throughout the universe. Open Access process eliminates the barriers associated with the older publication models, thus matching up with the rapidity of the twenty-first century. Muammoning dolzarbligi bo`lajak tarix fani o'qituvchilarining kasbiy va didaktik madaniyatini shakllantirish muammosi yetarlicha ishlab chiqilmaganligi bilan bog'liq. Bo`lajak tarix fani o'qituvchilari tarkibini takomillashtirish muammosini hal qilishga qaratilgan. Ushbu muammoni o'rganishda yetakchi yondashuv bo`lajak tarix fani o'qituvchilarining madaniy tarkibiy qismini ochib berishga imkon beradigan, kasbiy rivojlanishga tizimli, kompetensiyaga asoslangan, shaxsiy, aksiologik va boshqa yondashuvlarni o'z ichiga olgan madaniy yondashuv-didaktik mashg`ulot. Shuningdek, bo'lajak tarix fani o'qituvchilarining kasbiy va didaktik madaniyatining asosiy jihatlarini ko'rib chiqish va o'qituvchining individual elementlarini shakllantirish imkoniyatlarini tahlil qilishni talab etadi. Publishing centre of Finland 694 International Journal of Education, Social Science & Humanities. FARS Publishers Impact factor (SJIF) = 6.786 "Didactika" atamasi yunoncha "didactikos" so'zidan kelib chiqqan - o'qituvchi va "didasco" - o'quvchi. Ushbu konsepsiya birinchi marta nemis o'qituvchisi Volfgang Ratke tomonidan o'qitish san'atini ifoda etish uchun ilmiy muomalaga kiritilgan. Didaktika-bu fan va shu bilan birga o'qitish san'ati, tarixan mavjud bo'lgan madaniyatning muhim elementi rivojlangan. O'qituvchining faoliyati maqsad va vazifalar, hal qilish vositalari va usullari bilan belgilanadi. O'quv jarayonining o'zi rivojlangan madaniyat va ushbu davrda pedagogik jarayonni amalga oshirishda o'qituvchi faoliyatining usullari bilan belgilanadi. Ta'lim tizimida didaktika pedagogikaning o'quv jarayonini tashkil etishning nazariy asoslarini, uning qonuniyatlari, tamoyillari, madaniy tajribani o'zlashtirish usullarini ishlab chiqishga qaratilgan bo'lim sifatida ishlaydi. Y.K.Babanskiy pedagogik jarayonning tuzilishida aniqlangan: maqsad va vazifalar, o'qituvchilar va talabalar o'rtasidagi o'zaro munosabatlarning mazmuni, usullari, vositalari va shakllari, shuningdek, bu holda erishilgan natijalar. Bo'lajak tarix o'qituvchisining kasbiy tayyorgarligi turli va nisbatan mustaqil komponentlarni birlashtirgan dinamik tizimdir. Bo'lajak tarix fani o'qituvchilarining kasbiy-pedagogik shakllanishining ajralmas elementi ularning didaktik madaniyatini tizimli va izchil rivojlantirishga qaratilgan didaktik tayyorgarligi hisoblanadi. Didaktik madaniyat bo'lajak tarix fani o'qituvchilarining kasbiy faoliyati va samarali ta'lim uchun fundamental, qaytarilmas ahamiyatga ega O.A.Igumnov “Didaktik madaniyat-bu bo'lajak mutaxassisning kasbiy madaniyatini shakllantirish uchun samarali didaktik faoliyatni ta'minlaydigan va o'qituvchi tomonidan yangi pedagogik qadriyatlar va texnologiyalarni yaratish va rivojlantirishga hissa qo'shadigan o'qituvchi shaxsining ajralmas sifati", deb hisoblaydi. Bo'lajak tarix fani o'qituvchilarining didaktik madaniyatini rivojlantirish jarayoni bir nechta nazariy ko'rsatmalarga ega. Didaktik madaniyat kasbiy va pedagogik madaniyatning tarkibiy qismi sifatida bir xil o'ziga xos xususiyatlarga ega: elementlarni yoki tarkibiy qismlarni ularning birlashishiga asos bo'lgan ma'lum bir prinsip asosida ajratish; ushbu komponentlar o'rtasida ma'lum munosabatlar yoki munosabatlarning mavjudligi; ularning bir-biri bilan o'zaro ta'siri; tizimning ajralmas birlik sifatida ishlashi; tanlangan elementlarda muntazamlik mavjudligi. Ta'lim mazmuni anonim, umumiy, statik, ijtimoiy tajriba izomorfik, shaxssiz, kimdir tomonidan va bir vaqtning o'zida olingan. Ta'lim mazmuni shaxsiy, Publishing centre of Finland 695 International Journal of Education, Social Science & Humanities. FARS Publishers Impact factor (SJIF) = 6.786 samimiy, takrorlanmaydigan, "bu yerda va hozir" ochilmoqda. Darsning mazmuni aslida o'quv jarayonida yaratiladi va u bo`lajak tarix fani o'qituvchisining didaktik madaniy ijodining mahsuli sifatida ishlaydi. Xuddi shu mahsulot pedagogik jarayonning bevosita ishtirokchilarining munosabatlari, maqsadli munosabatlar, ularning motivlari, qiymat yo'nalishlari va hamkorlik usullari. Shuning uchun tizimli-kulturologik yondashuv doirasida "didaktik madaniyat" tushunchasini belgilashda biz bo`lajak tarix fani o'qituvchilarining didaktik faoliyati natijasida madaniy komponentning ustunligidan kelib chiqamiz. Bo'lajak tarix o'qituvchilarining didaktik madaniyati kasbiy kompetensiya, gumanistik shaxsiy pozitsiya, qadriyat yo'nalishlari tizimi, zamonaviy pedagogik fikrlash, rivojlangan ijodiy qobiliyatlar, o'qituvchining kasbiy va pedagogik madaniyatini o'z-o'zini rivojlantirish va bo`lajak tarix fani o'qituvchisi shaxsini rivojlantirishga asoslangan. Bu innovatsion va didaktik faoliyatda, bo'lajak tarix o'qituvchilarining ta'limi va shaxsiy rivojlanishida namoyon bo'ladigan ta'lim madaniyati o'qituvchisi tomonidan o'z imkoniyatlarini maksimal darajada amalga oshirishga qaratilgan tizimli sifatdir. Didaktika-bu ta'lim va tarbiya nazariyasi haqida tushuncha beradigan pedagogikaning bir bo'limi. Didaktikaning predmeti jarayon sifatida, tizim sifatida, shaxsni tarbiyalash va tarbiyalash vositasi sifatida, ya'ni o'qitish va ularning birligi va o'zaro bog'liqligi bilan o'zaro bog'liqligi, o'qituvchi tomonidan belgilangan talabalar tomonidan ta'lim mazmunini o'zlashtirilishini ta'minlaydi. Didaktik madaniyat-bu ta'lim madaniyati, ta'lim va shaxsiy rivojlanishga qaratilgan kasbiy va pedagogik faoliyat madaniyati. Bo`lajak tarix fani o'qituvchilarining didaktik madaniyati-bu uning shaxsiyatining ajralmas sifati, innovatsion o'qitish texnologiyalariga asoslangan didaktik faoliyat sohasida o'qituvchi sifatida ularning kasbiy va umumiy madaniy kompetentsiyasining umumiyligi, gumanistik va madaniy-tarixiy qadriyatlarni saqlash, bilimlar tizimi, qadriyat tendentsiyalari va o'quvchining shakllanishiga qo'shgan hissasi. Shuningdek, ijodiy o'z-o'zini rivojlantirish, o'qituvchining didaktik madaniyati quyidagi tarkibiy qismlardan iborat: - subyekt komponenti (shu jumladan, shaxsiy faoliyat, motivatsion qiymat, dizayn va texnologik elementlar); -subyekt komponenti (shu jumladan: vatanparvarlik, siyosiy savodxonlik, tanqidiy fikrlash, ijtimoiy-madaniy savodxonlik). Xulosa o'rnida aytishimiz mumkinki, zamonaviy ta'lim tizimida bo`lajak tarix fani o'qituvchilarining kasbiy va didaktik madaniyatini shakllantirish muammosini hal qilish zarurligini ko'rsatadi. Publishing centre of Finland 696 International Journal of Education, Social Science & Humanities. FARS Publishers Impact factor (SJIF) = 6.786 FOYDALANILGAN ADABIYOTLAR: 1. Атанов, Г. А. Ползучий дидактический нигилизм, или о деятельностном подходе в обучении [Текст] / Г. А. Атанов. // Образование и общество. - 2002. No 4. 2. Дидактическая культура преподавателя: Дидактический модуль [Текст]. - М.: Центроргтрудавтотранс, 2005. 3. Igamberdieva, K. E. (2022). Internal Audit in Business Subjects: Goals, Objectives and Principles. International Journal of Innovative Research in Science Engineering and Technology (IJIRSET), 11(3), 2322- 2330. 4. Игамбердиева, К. Э. (2020). Роль учѐта и контроля строительных материалов в строительной организации железнодорожного транспорта треста Мостострой. Вестник Хорезмской академии Маъмуна, 12, 83-84. 5. Джуманова, А. Б., Игамбердиева, К. Э. (2018). Последствия безработицы и их влияние на развитие экономики. Экономика и социум, 10, 243-245. Publishing centre of Finland 697 

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PHYSICAL REVIEW RESEARCH 2, 012049(R) (2020) Rapid Communications Abrupt phase transition of epidemic spreading in simplicial complexes Joan T. Matamalas , Sergio Gómez , and Alex Arenas Departament d’Enginyeria Informàtica i Matemàtiques, Universitat Rovira i Virgili, 43007 Tarragona, Spain (Received 7 October 2019; accepted 22 January 2020; published 27 February 2020) Recent studies on network geometry, a way of describing network structures as geometrical objects, are revolutionizing our way to understand dynamical processes on networked systems. Here, we cope with the problem of epidemic spreading, using the susceptible-infected-susceptible (SIS) model, in simplicial complexes. In particular, we analyze the dynamics of the SIS in complex networks characterized by pairwise interactions (links) and three-body interactions (filled triangles, also known as 2-simplices). This higher-order description of the epidemic spreading is analytically formulated using a microscopic Markov chain approximation. The analysis of the fixed point solutions of the model reveals an interesting phase transition that becomes abrupt with the infectivity parameter of the 2-simplices. Our results pave the way to advance in our physical understanding of epidemic spreading in real scenarios where diseases are transmitted among groups as well as among pairs and to better understand the behavior of dynamical processes in simplicial complexes. DOI: 10.1103/PhysRevResearch.2.012049 The collective behavior of dynamical systems on networks, has been a major subject of research in the physics community during the last decades [1–5]. In particular, our understanding of both natural and man-made systems has significantly improved by studying how network structures and dynamical processes combined shape the overall system behavior. Recently, the network science community has turned its attention to network geometry [6–9] to better represent the kinds of interactions that one can find beyond typical pairwise interactions. These higher-order interactions are encoded in geometrical structures that describe the different kinds of simplex structure present in the network: a filled clique of m + 1 nodes is known as an m-simplex, and together a set of 1-simplexes (links), 2simplexes (filled triangles), etc., comprise the simplicial complex. While simplicial complexes have been proven to be very useful for the analysis and computation in high dimensional data sets, e.g., using persistent homologies [10–14], little is understood about their role in shaping dynamical processes, save for a handful of examples [15–18]. A more accurate description of dynamical processes on complex systems necessarily requires a new paradigm where the network structure representation helps to include higherorder interactions [19]. Simplicial geometry of complex networks is a natural way to extend many-body interactions in complex systems. The standard approach so far consists in understanding the coexistence of two-body (link) interactions and three-body interactions (filled triangles). Note that this approach is different from considering pairwise interactions 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. 2643-1564/2020/2(1)/012049(5) 012049-1 among three elements of a triangle, it refers to the interaction of the three elements, in the filled triangle, at unison. Here we present a probabilistic formalization of the higherorder interactions of an epidemic process, represented by the well-known susceptible-infected-susceptible (SIS) model [20] in one- and two-simplices, revealing that the consideration of higher-order structure (filled triangles) can change the character of the phase transition of the epidemic spreading to the endemic state. Specifically, we find that for a significant region of the parameters space the continuous phase transition that has been well-characterized in networks so far [5] becomes abruptly discontinuous [21]. These results are important for physicists working on network science, independently on the particular dynamical process we have chosen, to explain why this physical phenomena could arise in complex systems. Similar results have been reported for social contagion dynamics [17] using a mean-field approach. For the mathematical formalization of the dynamical process, we use simplicial complexes extensions of the microscopic Markov chain aproach (MMCA) [22–27], and of the epidemic link equations (ELE) [28], that compute the probabilities of 1 and 2-simplexes to transmit the epidemics. This formalism allows us to get physical insight into the phase transition and its consequences at the level of our understanding of plausible epidemic scenarios. Let us start by considering the dynamics of the SIS model in networks. We consider a network of N nodes and L links, where the nodes can have two states, susceptible (S) or infected (I). The classical interaction so far considers that the infection propagates, pairwise, from certain infected individuals to their neighbors with a probability β, and infected nodes eventually recover with probability μ. In the simplicial complex scenario, we will consider also triangular interactions, i.e., every node also interacts within the 2-simplexes with the two neighbors at unison, with an infection probability β  when the other two members are infected. We can define a system of N discrete-time MMCA equations that capture the Published by the American Physical Society MATAMALAS, GÓMEZ, AND ARENAS PHYSICAL REVIEW RESEARCH 2, 012049(R) (2020) evolution over time of the probability pi of node i being in an infected state as (a) pi (t + 1) = (1 − pi (t ))(1 − qi (t )qi (t )) + pi (t )(1 − μ) , (1) 0.6 where qi (t ) defines the probability that node i is not infected by any pairwise interaction with its neighbors,  (1 − β pi (t )) , qi (t ) = (2) 0.4 0.2 j∈i 0.0 10−2 10−1.5 ρ and qi (t ) is the probability that node i is not infected by any of its interactions at the 2-simplicial level,  qi (t ) = (1 − β  p j (t )p (t )) . (3) 10−1 (b) 0.8 j,∈i Here, i and i represent the sets of 1 and 2-simplexes containing node i, respectively. The system of equations (1) updates the probability of a node i being infected as the probability of being susceptible at time t and becoming infected by some neighbor, or some group of neighbors in a triangle [first term on the right-hand side (r.h.s.) of the equation], or the probability that node i was already infected at time t and it does not recover. This system of equations is a contraction map Tβ,β  ,μ : p(t ) → p(t + 1) for every value of the parameters, and then the existence of fixed points is guaranteed by the Banach fixed point theorem [29]. We can solve the system by iteration. A naïve approximation of Eq. (1) is the homogeneous assumption in which all nodes have the same degree (k), belong to the same number of 2-simplexes (k  ), and have the same probability of being infected (pi (t ) = p(t )). Expanding up to second order in p, and developing the equation at the stationary state (p(t ) = p), it reveals the structure responsible for the abrupt transitions we could foresee: (1 − p)[βk p + (β  k  − β 2 k(k − 1)/2)p2 ] − μp = 0 . (4) This third-order algebraic equation has a trivial solution, p = 0, and two more roots, that depending on the parameters, provides up to two additional physical solutions with p ∈ (0, 1]. For the parameters with three physical solutions, the stability analysis shows that the middle one is unstable, thus being responsible of the abrupt transitions, see Fig. 1. This result is equivalent to that obtained in [17]. Note, however, that Eq. (1) and in turn Eq. (4) carries the implicit assumption that the probability of being infected by one neighbor is independent on the probability of being infected by any other neighbor. This assumption is a meanfield approximation, whose validity is severely compromised in the current scenario, where we account for the infectivity in triads, and hence neighbors are unlikely to be independent. To palliate the previous limitation, we can define the simplicial epidemic model at a level of links using a system of 3L equations. The size of this system is very large compared to the previous MMCA system of N equations at the level of nodes; however, we can simplify the model to a system of L + N equations if we consider the constrains imposed by the probabilistic model, i.e., the node marginal probabilities have to be the same regardless the link that we consider to compute them. These restrictions read as follows: for a 0.6 0.4 0.2 0.0 10−2 10−1.5 10−1 β FIG. 1. Phase diagram of the mean-field approximation detailed in Eq. (4). We consider a network with average degree k = 12, an average number of triangles that each node is part of k   = 5, a recovery probability μ = 0.2, and two different infection probabilities through ternary interactions: (a) β  = 0.0; (b) β  = 0.1. Solid lines represent stable solutions and dashed lines depict unstable solutions to Eq. (4). The dotted area represents the region of parameters that enables a convergence to an endemic stable state from below. link connecting nodes i and j, the probability of a node i SI SS to be in the susceptible state is PiS = PiSS j + Pi j , where Pi j is the joint probability of node i being susceptible and node j being susceptible, and PiSIj is the joint probability of node i being susceptible and node j being infected. Equivalently PiI = PiIIj + PiISj . Wrapping up these restrictions we can write the epidemic link equations, ELE [28], for the simplicial model, for every node i, as    PiI (t + 1) = 1 − PiI (t ) 1 − qi (t )qi (t ) + PiI (t )(1 − μ) , (5) where qi (t ) defines the probability that node i is not infected by any pairwise interaction with its neighbors,    PiSIj (t ) 1−β S qi (t ) = , (6) Pi (t ) j∈ i qi (t ) is the probability that node i is not infected by any and of the interactions at the 2-simplicial level,   SII  P (t ) i jr  1 − β S qi = . (7) Pi (t ) j,r∈ i Note that, to write down the previous equations, we have made use of Bayes’ theorem, substituing the conditional 012049-2 ABRUPT PHASE TRANSITION OF EPIDEMIC SPREADING … PHYSICAL REVIEW RESEARCH 2, 012049(R) (2020) (a) 0.8 0.6 0.4 j i 0.2 0.0 10−1 ρ 10−2 100 (b) 0.8 0.6 β SI (t) Pir PiS (t) β SII Pir (t) PiS (t) β SII Pijr (t) FIG. 2. Schematic representation of the contribution of different interactions to the joint probability between the states of node i and node j, through node i. Black solid lines account for direct interactions between node i and its neighbors. The checked pattern area represents a 2-simplex interaction with node i where node j is not participating, and the dotted pattern areas represent 2-simplex interactions with node i where node j is participating. All these contributions participate in Eqs. (9) to (11), respectively. probabilities PjiI|S of node j to be infected knowing that node i is susceptible, by the joint probability PiSIj /PiS ; equivalently, II|S S = PiSII for the 2-simplicial terms, Pjri jr /Pi . The system still requires of L equations, one for every link, that account for the probability of having a link connecting two nodes in the infected state II, transitioning from the four possible states SS, SI, IS, II. It reads      PiIIj (t + 1) = PiSS j (t ) 1 − qi j (t )qi j (t ) 1 − q ji (t )q ji (t )   + PiSIj (t ) 1 − (1 − β )qi j (t )uij (t )qij (t ) (1 − μ)   + PiISj (t ) 1 − (1 − β )q ji (t )uji (t )qji (t ) (1 − μ) + PiIIj (t )(1 − μ)2 , (8) where we have used: the probability of node i not being infected by any neighbor different from j through a link,   PirSI (t ) 1−β S ; qi j (t ) = Pi (t ) r∈i (9) r= j the probability of node i not being infected by any 2-simplex not containing node j, qij (t )  SII   Pir (t ) 1−β ; = PiS (t ) r,∈ r= j = j i 0.4 PijSI (t) (10) ELE MMCA MF 0.2 0.0 10−2 10−1 100 β FIG. 3. Incidence of the epidemic ρ as function of infection probability β on a random network of N = 2000 nodes. We consider a network with average degree k = 12, an average number of triangles that each node is part of k   = 5, a recovery probability μ = 0.2, and two different infection probabilities through ternary interactions: (a) β  = 0.0 and (b) 0.1. The incidence has been analytically computed using ELE (solid line), MMCA (dashed line) and the mean-field approximation (dotted line). Results obtained from Monte Carlo simulations, performed using the quasistationary approach [33], are depicted by solid dots. and the probability of node i not being infected by any 2simplex contatining node j,    PSII (t )   i jr ui j (t ) = 1−β . (11) PiSIj (t ) r∈ ij See an illustration of the different contributions to Eqs. (9) to (11) in Fig. 2. To solve the system of Eqs. (5) and (8), we still need a closure for the ternary joint probabilities PiSII jr found in Eqs. (10) and (11). To break the hierarchy of clusters produced by these terms we must rely on approximations. The classical pair approximation in statistical physics [30], also used in the context of epidemics by Mata and Ferrerira [31], SI II I would consist in approximating PiSII jr ≈ Pi j Pjr /Pj . However, Cator and Van Mieghem [32], propose a different closure SI I PiSII jr ≈ Pi j Pr . The main problem with these proposals in the current scenario is that the symmetry of the elements in the 2-simplexes is broken, and this concurs in a degeneration of solutions. For example, using the second option, we could SI I SI I choose between three different closures for PiSII jr : Pi j Pr , Pir Pj , II S or Pjr Pi . Each alternative makes use of one node and one link (the link opposite to the chosen node in the triangle) probabilities, but there is no indication in the structure of 012049-3 MATAMALAS, GÓMEZ, AND ARENAS PHYSICAL REVIEW RESEARCH 2, 012049(R) (2020) (a) 0.8 0.6 0.4 0.2 0.0 10−4 10−3 10−2 10−1 100 (b) 0.8 0.6 0.4 ELE MMCA MF 0.2 0.0 10−4 10−3 10−2 10−1 100 FIG. 4. Incidence of the epidemic ρ as function of infection probability β on a random network with N = 8000 nodes, built according to [34]. The average degree of the network is k = 4 and the average number of triangles per node is k   = 3. The recovery probability is μ = 0.2, and we use two different infection probabilities through ternary interactions: (a) β  = 0.00 and (b) 0.14. The incidence has been analytically computed using ELE (solid line), MMCA (dashed line) and the mean-field approximation (dotted line). Results obtained from Monte Carlo simulations, performed using the quasistationary approach [33], are depicted by solid dots. which ones should be preferred. To avoid such degeneracies, the closure proposal should be symmetric, and hence our approach consists in the following closure approximation: connected triads of nodes, not necessarily forming triangles as in our current case. The results of the previous mathematical formulation can now be obtained by fixed point iteration. In Fig. 3, we present the results for homogeneous random networks, when the simplicial structure is not considered (β  = 0), and when 2-simplices are included (β  = 0). We observe that in the second case, when the simplical structure is considered for the higher-order dynamics of the SIS model, the incidence of the epidemics ρ = N1 i PiI reveals an abrupt transition in all the previous approaches: MMCA, its mean-field version MF, and ELE. Nevertheless, the most accurate approximation when compared with the Monte Carlo simulation of the system, is provided by the epidemic link equations ELE. This is specially crucial when we try to capture the critical point of the transition. Unfortunately, the critical point at which the transition occurs eludes our analytical determination in ELE, given that nor the usual linearization technique [22,28], neither the next generation matrix method (NGM) [35] provided meaningful results. In Fig. 4, we corroborate our results on heterogeneous (scale-free) networks constructed following the analytical proposal in Ref. [34]. We observe how heterogeneity in degree highlights the differences in the determination of the critical point obtained by ELE, MMCA, and MF. Summarizing, we have presented the mathematical formulation of the SIS model in simplicial complexes, using a discrete time probabilistic description of the process, in the node approximation MMCA, and in the link approximation ELE. Both descriptions predict an abrupt transition, as well as the stationary homogeneous approximation of the MMCA, the MF. The accuracy of the predictions is largely better for ELE, and reveals that this approximation is extremely useful when dealing with the simplicial geometry of complex networks. For the determination of the critical points of ELE, we think that further analysis using ideas on stability of subsystems [36] of 1-simplices and 2-simplices, are a promising line of research. The results are not only important for epidemic spreading, but for any other contagion process that can be described within the probabilistic framework of MMCA. In this way, all the node and link probabilities of the 2-simplex structures are used, avoiding the asymmetries introduced in the two previous closures, which were designed to handle We acknowledge support by Ministerio de Economía y Competitividad (Grants No. PGC2018-094754-B-C21 and No. FIS2015-71929-REDT), Generalitat de Catalunya (Grant No. 2017SGR-896), and Universitat Rovira i Virgili (Grant No. 2018PFR-URV-B2-41). A.A. acknowledges also ICREA Academia and the James S. McDonnell Foundation (Grant No. 220020325). [1] S. H. Strogatz, Sync: The Emerging Science of Spontaneous Order (Hypernion, 2003). [2] A. Arenas, A. 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www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 33), pp: 54388-54401 Research Paper ZEB1 confers stem cell-like properties in breast cancer by targeting neurogenin-3 Chen Zhou1,*, Huimin Jiang1,*, Zhen Zhang1, Guomin Zhang1, Hang Wang1, Quansheng Zhang2, Peiqing Sun3, Rong Xiang1 and Shuang Yang1 1 Tianjin Key Laboratory of Tumor Microenvironment and Neurovascular Regulation, Medical School of Nankai University, Tianjin 300071, China 2 Tianjin Key Laboratory of Organ Transplantation, Tianjin First Center Hospital, Tianjin 300192, China 3 Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA * These authors have contributed equally to this work Correspondence to: Shuang Yang, email: yangshuang@nankai.edu.cn Keywords: breast cancer, neurogenin-3, stemness properties, tumor initiation, ZEB1 Received: December 29, 2016     Accepted: March 20, 2017     Published: April 13, 2017 Copyright: Zhou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Cancer stem cells (CSCs) are a subpopulation of cancer cells believed to be implicated in cancer initiation, progression, and recurrence. Here, we report that ectopic expression of zinc finger E-box binding homeobox 1 protein (ZEB1) results in the acquisition of CSC properties by breast cancer cells, leading to tumor initiation and progression in vitro and in vivo. The neurogenin 3 gene (Ngn3) is a bona fide target of ZEB1, and its repression is a key factor contributing to ZEB1-induced cancer cell stemness. ZEB1 suppressed Ngn3 transcription by forming a ZEB1/DNA methyltransferase (DNMT)3B/histone deacetylase 1 (HDAC1) complex on the Ngn3 promoter, leading to promoter hypermethylation and gene silencing. The rescue of Ngn3 expression attenuated ZEB1-induced cancer stemness and symmetric CSC division. Immunohistological analysis of human breast cancer specimens revealed a strong inverse relationship between ZEB1 and NGN3 protein expression. Thus, our findings suggest ZEB1-mediated silencing of Ngn3 is required for breast tumor initiation and maintenance. Targeted therapies against the ZEB1/Ngn3 axis may be highly valuable for the prevention and treatment of breast cancer. characteristics. Thus, this process results in simultaneous differentiation and self-renewal, while maintaining the number of stem cells constant for tissue homeostasis [7]. Symmetric cell division, in contrast, generates only stem cells. Whereas the resulting expansion of the stem cell population could be useful for tissue regeneration under normal physiological conditions, the deregulation of asymmetric and/or symmetric division may, under pathogenic conditions, promote tumor initiation [8]. For example, loss of tumor suppressor genes such as the adenomatous polyposis coli gene (APC), often favors increased symmetric division of CSCs, potentially leading to tumorigenesis [9]. Therefore, understanding the complex regulatory network that interconnects cell polarity and cell fate is paramount to elucidate the role of INTRODUCTION Although cancer stem cells (CSCs) constitute just a small fraction of the total tumor cell population, they are thought to be the main drivers of tumorigenesis and cancer recurrence [1, 2]. There is evidence that CSC numbers correlate well with aggressive tumor growth [3, 4]; moreover, specific changes in the microenvironment may induce, through mutagenic events or epigenetic alterations, a CSC expansion that potentiates tumor progression [5, 6]. Current knowledge indicates that CSC properties may be largely determined by evolutionarily conserved mechanisms dictating cell polarity and asymmetric cell division. Normal CSCs usually undergo asymmetric cell division, giving rise to daughter cells with different www.impactjournals.com/oncotarget 54388 Oncotarget CSCs in carcinogenesis and to effectively guide preventive and therapeutic efforts. Zinc finger E-box binding homeobox 1 (ZEB1), a pivotal member of the zinc finger-homeodomain transcription factor family, importantly influences developmental and homeostatic cell fate decision in a broad range of tissues [10–12]. The overexpression of ZEB1, demonstrated in breast, colon, prostate, and pancreatic cancer [13–16], contributes to impaired cell adherence and polarity [17], induction of epithelial to mesenchymal transition (EMT) [18], and the acquisition of chemo- and radio-resistant phenotypes [19, 20]. A growing body of evidence suggests that ZEB1 promotes the generation of breast CSCs [21–23]. For instance, elevated expression of ZEB1 predicts radiotherapy relapse in triple-negative breast cancer patients, in which the CSC population is enlarged [20]. Moreover, ectopic expression of ZEB1 in breast cancer is readily influenced by microenvironmental signals such as TGF-β, resulting in CSCs population expansion by non-CSCs to CSCs conversion and increased tumorigenesis [24]. Interestingly, ZEB1-related stemness properties have been shown to arise rather indirectly, through the transcriptional regulation of miRNAs such as miR-200 and miR-203 [18]. However, the cellular and molecular mechanisms downstream of ZEB1 that contribute to tumorigenesis remain largely obscure. In this study, we provide evidence that ZEB1 promotes CSC self-renewal in vitro and in vivo, thus leading to increased breast cancer initiation and growth. By reduced representation bisulfite sequencing (RRBS) analysis, we identified neurogenin-3 (Ngn3) as a bona fide target of ZEB1 implicated in the acquisition of cancer cell stemness. ZEB1 represses Ngn3 transcription by recruiting DNA methyltransferase (DNMT)3B and histone deacetylase (HDAC)1 to the Ngn3 promoter, thus resulting in DNA hypermethylation and the silencing of Ngn3. We also demonstrated an inverse correlation between ZEB1 and NGN3 expression in human breast cancer specimens. Notably, rescuing Ngn3 expression significantly attenuated ZEB1-induced CSC self-renewal and stemness. Our data elucidates an important role for ZEB1/Ngn3 signaling in regulating stem cell fate and suggests a potential therapeutic target for ZEB1-overexpressing cancers. (Figure 1A and Supplementary Figure 1B). Flow cytometry and immunohistochemical staining analysis further revealed increased activity of ALDH, a malignant human mammary stem cell marker [21], in tumors from ZEB1/231 xenografts (Figure 1B and Supplementary Figure 1C). In addition, measurement of the CD44+CD24− breast CSC population also demonstrated that ectopic expression of ZEB1 led to increased stemness properties in xenografted MDAMB-231 breast cancer cells (Figure 1C). Next, by measuring three well-established CSC endpoints, i.e. tumorsphere formation, side population (SP) percentage, and stemness-related gene expression, we conducted in vitro studies to confirm that ZEB1 targets, in fact, breast CSCs. Our results demonstrated that ectopic ZEB1 expression in MDA-MB-231 cells increased tumorsphere formation (Figure 1D), SP percentage (Figure 1E), and the expression of pluripotency markers such as NANOG, OCT4, and SOX2 (Figure 1F). Additionally, we explored the effect of ZEB1 on CSC polarity during division by assessing, shortly after mitosis, the subcellular distribution of NUMB, a protein involved in cell fate specification [25]. To this end, cell division symmetry was evaluated in ZEB1/231 and Ctrl/231 cells treated with blebbistatin, a cytokinesis-arresting small molecule that induces formation of binucleated cells (Figure 1G). Results showed that CD44 expression was abolished in up to 64.7% of mitotic Ctrl/231 cells, while NUMB was uniformly distributed around the actin cortex (Figure 1G and 1H), suggesting that both daughter cells lost their stem cell identity upon symmetric cell division (symmetric commitment [SC]). In 27.3% of the Ctrl/231 cells, CD44 and NUMB were asymmetrically distributed [asymmetric division [AD]), wherein NUMB was present only in CD44progeny, but not in CD44+, stem cell-like cells (Figure 1G and 1H). In up to 8.0% of the binucleated Ctrl/231 cells, on the other hand, CD44 expression was symmetric, and NUMB showed a uniform distribution, thus manifesting a symmetric division (SD) phenotype. Interestingly, as shown in Figure 1H, this SD phenotype was substantially enhanced after ectopic expression of ZEB1 (8.0% to 32.1%). In contrast, ZEB1 depletion achieved the opposite effect, i.e. reduced cancer stem-like cell properties (Supplementary Figure 2). These observations were not unique to MDAMB-231 cells; ZEB1 overexpression or depletion in SUM159 cells also resulted in enhanced and reduced CSC phenotypes, respectively (Supplementary Figure 3 and 4). In addition to the results from the previous study indicating that ZEB1 is capable of inducing EMT [26], the present data also suggest that ZEB1 is crucial for the acquisition or conservation of stemness properties in breast CSCs. RESULTS ZEB1 induces stemness properties in breast cancer cells To assess the influence of ZEB1 expression on breast cancer initiation in vivo, we performed extreme limiting dilution analysis to detect ZEB1-induced CSC frequencies. To this end, ZEB1 gain-of-function transfection was performed in MDA-MB-231 breast cancer cells to establish a nude mouse xenograft model (Supplementary Figure 1A). Compared with control (Ctrl/231) tumor cells, ZEB1/231 tumor cells displayed a 32.8-fold increase in CSC frequency www.impactjournals.com/oncotarget ZEB1 downregulates Ngn3 via promoter hypermethylation Based on mounting evidence implying a contribution of ZEB1 to epigenetic regulation during tumorigenesis [27], we performed RRBS to identify endogenous targets 54389 Oncotarget Figure 1: ZEB1 induces stemness properties in breast cancer cells. (A) For in vivo limited dilution assays, a total of 2×106, 1.0×106, 5×105, 2.5×105, and 1.25×105 ZEB1/231 or Ctrl/231 cells were injected into the mammary fat pads of nude mice. After 15 days, the mice were euthanized, and estimated CSC frequency was analyzed using ELDA software. Data were analyzed with chi-square tests (*P = 0.097). (B) and (C) Tumor tissues were prepared in single-cell suspension and processed for ALDH activity (B) and CD44+CD24population (C) analysis by flow cytometry. *P < 0.05, **P < 0.01 vs. the respective control by Student’s t-test. (D) ZEB1/231 or Ctrl/231 cells were prepared in single-cell suspension and seeded in 24-well ultra-low attachment plates at 1,000 cells/well. Tumorsphere formation was examined after 14 days of culturing. ***P < 0.001 vs. the respective control by Student’s t-test. (E) ZEB1/231 or Ctrl/231 cells were stained with Hoechst 33342 in the presence or absence of verapamil. The side population fraction was examined by flow cytometry. ***P < 0.001 vs. the respective control by Student’s t-test. (F) The full-length ZEB1 expression plasmid was introduced into MDAMB-231 cells. At the indicated time points, the expression of ZEB1, NANOG, OCT4, and SOX2 was assessed by immunoblotting and was normalized to the levels of β-Actin. (G) Schematic showing SD, AD, and SC patterns. (H) Confocal fluorescence images showing CD44 (red) and NUMB (green) intracellular distribution during the first cell division of the CD44+CD24- population isolated from ZEB1/231 and Ctrl/231 cells and cultured in suspension with blebbistatin treatment. The percentage of the SD versus AD populations was analyzed. *P < 0.05, ***P < 0.001 vs. the respective control by Student’s t-test. Scale bars: 20 μm. www.impactjournals.com/oncotarget 54390 Oncotarget Figure 2: The Ngn3 promoter is a bona fide hypermethylation target of ZEB1. (A) Schematic representation of RRBS identification of the putative ZEB1 targets involved in breast tumorigenesis. Quantitative PCR were performed to verify the downregulation of MerTK, Cyp26B1, EphA2, and Ngn3 expression in ZEB1/231 vs. Ctrl/231 cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the respective control by Student’s t-test. (B) The downregulation of NGN3 protein expression was assessed by immunoblotting in ZEB1/231 vs. Ctrl/231 cells and normalized to the levels of β-Actin. (C) and (D) The percentage of DNA methylation of the Ngn3 promoter was determined by BSP in ZEB1/231 vs. Ctrl/231 cells (C) and in shZEB1/231 vs. shCtrl/231 cells (D). **P < 0.01, ***P < 0.001 vs. the respective control by Student’s t-test. (E) and (F) ZEB1/231 and Ctrl/231 cells were treated with AZA (1.5 μM) and TSA (2 mM) for the indicated time points. The expression of Ngn3 was assessed by quantitative PCR (E) and immunoblotting (F) and normalized to the levels of β-Actin. ***P < 0.001 vs. the respective control by Student’s t-test. www.impactjournals.com/oncotarget 54391 Oncotarget (Figure 2A). Considering that Ngn3 influences stem cell properties and cell differentiation [28], immunoblotting was performed to verify that a negative correlation existed between ZEB1 and NGN3 protein expression (Figure 2B). Thus, these data indicate that ZEB1 promotes CSC renewal through repression of Ngn3 expression. To further confirm that the inhibition of Ngn3 expression by ZEB1 in breast cancer is correlated with DNA methylation, we conducted bisulfite sequencing PCR (BSP) to evaluate the methylation status of 13 CpG residues in the 243-bp DMR region (-164/+79) of the Ngn3 promoter (Supplementary Figure 5A). Relative to of ZEB1 in MDA-MB-231 cells. We identified 291 genes containing differentially methylated regions (DMRs) in ZEB1/231 cells, as compared with Ctrl/231 cells. 22 of these genes possess potential tumorigenic functions and contain E2-box motifs for ZEB1 binding in their promoter regions (Figure 2A). We then performed quantitative PCR to determine the correlation between ZEB1 and these differentially methylated genes in ZEB1/231 cells. The results showed that ectopic ZEB1 expression significantly downregulated the mRNA levels of MerTK (c-mer protooncogene tyrosine kinase), Cyp26B1 (Cytochrome P450 26B1), EphA2 (ephrin type-A receptor 2), and Ngn3 Figure 3: ZEB1 represses Ngn3 transcription via interaction with DNMT3B and HDAC1. (A) MDA-MB-231 cells were co-transfected with the ZEB1 expression plasmid and different wild-type or truncated Ngn3 promoter luciferase reporter constructs. Cell extract luciferase activities were determined 36 h after transfection using a Betascope analyzer. Luciferase values were normalized to Renilla activities. ***P < 0.001 vs. the respective control by Student’s t-test. (B) The overexpression of ZEB1 significantly enhanced its recruitment to the endogenous Ngn3 promoter, as confirmed by a quantitative ChIP assay using E2-box-specific primers. ***P < 0.001 vs. the respective control by Student’s t-test. (C) The interactions among ZEB1, DNMT3B, and HDAC1 protein were analyzed by coimmunoprecipitation in ZEB1/231 cells. (D) and (E) ZEB1 overexpression significantly enhanced the recruitment of DNMT3B (D) and HDAC1 (E) to the endogenous Ngn3 promoter, as confirmed by a quantitative ChIP assay using E2-box-specific primers. ***P < 0.001 vs. the respective control by Student’s t-test. (F) MDA-MB-231 cells were co-transfected with the Ngn3-p-0.7k reporter and the ZEB1 expression plasmid, and then treated with AZA and TSA. Cell extract luciferase activities were determined 36 h after transfection using a Betascope analyzer. Luciferase values were normalized to Renilla activities. *P < 0.05, **P < 0.01 vs. the respective control by Student’s t-test. www.impactjournals.com/oncotarget 54392 Oncotarget to the Ngn3 promoter in an E2-box-3-dependent manner, and the association was further increased by ZEB1 overexpression (Figure 3D and 3E). Next, the ZEB1 expression plasmid and an Ngn3 promoter construct containing E2-box-3 were introduced into MDA-MB-231 cells, followed by treatment with AZA and TSA. Luciferase assay results showed that exposure to AZA and TSA significantly attenuated ZEB1-mediated repression of Ngn3 promoter activity (Figure 3F). These observations highlight the importance of the E2-box elements, especially E2-box-3, in the regulation of Ngn3 expression by ZEB1 via interaction with DNMT3B and HDAC1. Ctrl/231, DNA methylation was increased in ZEB1/231 cells (Figure 2C and Supplementary Figure 5B), while ZEB1 knockdown correlated instead with decreased DNA methylation (Figure 2D and Supplementary Figure 5C). Ngn3 mRNA expression was then compared by quantitative PCR in Ctrl/231 vs. ZEB1/231 cells and in shCtrl/231 vs. shZEB1/231 cells (Supplementary Figure 5D and 5E), confirming a negative regulation of Ngn3 expression by ZEB1. Gene regulation via promoter methylation is sometimes accompanied by an increase in the activities of DNMT and HDAC [29]. Thus, ZEB1/231 cells were treated with the demethylating agent 5-aza-2'deoxycytidine (AZA) and the HDAC inhibitor trichostatin A (TSA). These treatments significantly abolished ZEB1mediated downregulation of Ngn3 at the mRNA and protein levels (Figure 2E and 2F). We also examined ZEB1-regulated promoter methylation of Ngn3 in SUM159 cells and obtained similar results (Supplementary Figure 6). ZEB1 confers breast CSC properties by targeting Ngn3 Next, we tested whether ZEB1/Ngn3 signaling would functionally alter the stemness properties of MDA-MB-231 cells. Thus, a control- or Ngn3-expressing plasmid was introduced into ZEB1/231 cells. Rescue of Ngn3 expression was assessed by immunoblotting (Figure 4A). Measurements of tumorsphere formation, SP percentage, CD44+CD24− breast CSC population, ALDH activity, and expression analysis of pluripotency genes demonstrated that ectopic ZEB1 expression led to increased stemness properties, whereas these changes were significantly attenuated by re-expression of Ngn3 (Figure 4A-4E). Notably, as shown in Figure 4F, we observed that the SD phenotype was less represented (32.0% vs 13.3%), and the AD phenotype was enhanced (17.0% vs 31.3%), after ectopic expression of Ngn3 in ZEB1/231 cells; this demonstrated that ZEB1 is linked to stem cell identity in breast cancer cells, thus at least partially accounting for the downregulation of Ngn3 expression. We also examined the involvement of Ngn3 in ZEB1-regulated cancer cell stemness properties in SUM-159 cells, with similar results (Supplementary Figure 8). ZEB1 represses Ngn3 transcription by recruiting HDAC1 and DNMT3B to its promoter Next, we performed promoter-reporter assays to elucidate the molecular mechanism by which ZEB1 regulates Ngn3 transcription. As shown in Figure 3A, the wild-type -1702/+174 promoter of the Ngn3 gene has three canonical E2-box elements [CA(G/C)(G/C)TG] at positions -1308/-1303, -803/-798 and -517/-512, to which ZEB1 may potentially bind [30]. The luciferase assay results indicated that ZEB1 overexpression decreased the promoter activity of the Ngn3-p-1.7k reporter by approximately 68% relative to control MDA-MB-231 cells without ZEB1 transfection (Figure 3A). A series of truncated Ngn3 promoter-reporter constructs were then generated for analysis. The luciferase assays showed that the deletion of the -1702/-700 fragment, containing E2box-1 and -2, did not affect ZEB1-mediated repression of Ngn3 promoter activity. However, this transcriptional repression was eliminated after simultaneous deletion of all three E2-box elements. Importantly, quantitative ChIP assays indicated that ZEB1 overexpression resulted in a 20-fold increase in its binding to the area of E2-box-3, whereas the recruitment of ZEB1 to E2-box-1 and -2 was less evident (Figure 3B and Supplementary Figure 7). These observations suggest a predominant role of the E2box-3 element in the regulation of Ngn3 transcription by ZEB1. DNMT3B and HDAC1 have been shown to mediate the epigenetic regulatory functions of ZEB1 by acting as its cofactors [27]. We therefore performed co-immunoprecipitation experiments, which proved the interaction between ZEB1 and DNMT3B/HDAC1 in ZEB1/231 cells (Figure 3C). ChIP experiments further revealed that both DNMT3B and HDAC1 were recruited www.impactjournals.com/oncotarget Ngn3 is required for the induction of ZEB1mediated CSC properties in vivo Consequently, we tested whether ZEB1-regulated Ngn3 expression would alter breast CSC frequencies in vivo. To do so, re-expression of Ngn3 was performed in ZEB1/231 cells to establish a nude mouse xenograft model. The results of the extreme limiting dilution assay showed that rescuing Ngn3 expression in ZEB1/231 tumors significantly attenuated CSC frequency (Figure 5A and Supplementary Figure 9). Immunohistochemical staining and flow cytometry analysis further revealed that the increases observed in both ALDH activity and the CD44+CD24− CSC subpopulation in ZEB1/231 tumors were significantly abolished after re-expression of Ngn3 (Figure 5B to 5D). Altogether, these data suggest that Ngn3 is a ZEB1 target that may be functionally implicated in the acquisition of stem cell characteristics. 54393 Oncotarget Differential correlation of ZEB1 with NGN3 and ALDH1 expression in breast cancer specimens of their ZEB1 expression scores. The results indicated a strong negative correlation between the expression of ZEB1 and NGN3 (Figure 6A and 6B). Importantly, in tumors with high ALDH1 activity, we observed increased expression of ZEB1 and decreased expression of NGN3 (Figure 6C and 6D), strongly suggesting that dysregulated ZEB1/NGN3 signaling is functionally linked to tumor stem cell traits in breast cancer. To further strengthen the pathological correlation between ZEB1/Ngn3 expression and breast cancer cell stemness, we performed immunohistochemical staining for ZEB1, NGN3 and ALDH1 in 156 primary breast carcinoma patient specimens. As shown in Figure 6A, the subjects were divided into three groups on the basis Figure 4: ZEB1/Ngn3 signaling affects breast CSC properties in vitro. (A) ZEB1/231 cells were transfected with an Ngn3 expression plasmid. The expression of ZEB1, NGN3, NANOG, OCT4, and SOX2 was assessed by immunoblotting and normalized to the levels of β-Actin. (B) to (F) Ngn3-mediated ZEB1 regulation of CSC properties was determined by analyzing tumorsphere formation (B), side population (C), CD44+CD24- population (D), ALDH activity (E), and SD population frequency (F). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the respective control by Student’s t-test. www.impactjournals.com/oncotarget 54394 Oncotarget DISCUSSION We also assessed ZEB1 and NGN3 co-expression in tumors with different grades. The results revealed that ZEB1 expression was relatively lower (Figure 6E), while NGN3 expression was higher (Figure 6F) in low-grade tumors, a result consistent with findings from a previous report showing that cancer stem cells are enriched in highgrade tumors [25]. The presence of radiation- and chemotherapyresistant breast CSC populations contributing to tumor growth by promoting angiogenesis, tissue invasion, and metastasis is supported by multiple evidence. Therefore, the elucidation of molecular regulators of breast CSCs Figure 5: ZEB1/Ngn3 signaling affect breast CSC properties in vivo. (A) A total of 2×106, 1.0×106, 5×105, 2.5×105, and 1.25×105 ZEB1/231 cells with or without Ngn3 re-expression were injected into the mammary fat pads of nude mice. After 15 days, the mice were euthanized, and the estimated CSC frequency was analyzed using ELDA software. Data were analyzed with chi-square tests (*P = 0.0063). (B) The expression of ZEB1, NGN3, and ALDH1 in breast cancer xenografts was examined by immunohistochemical staining. Scale bars, 50μm. **P < 0.01, ***P < 0.001 vs respective control in Student’s t-test. (C) and (D) Tumor tissues were prepared in single-cell suspension and processed for ALDH activity (C) and CD44+CD24- population (D) analysis by flow cytometry. **P < 0.01, ***P < 0.001 vs. the respective control by Student’s t-test. www.impactjournals.com/oncotarget 54395 Oncotarget may translate into improved anti-neoplastic therapies. Considering our findings, we propose that ZEB1 is a key regulator of breast CSCs and may be critically involved in tumor initiation. First, we showed that ZEB1 is required for tumor initiation and expression of stemness markers in vitro and in vivo, and this effect is Ngn3-dependent. Second, we found that ZEB1 interacts with DNMT3B and HDAC1 at the Ngn3 promoter, thus leading to DNA hypermethylation and downregulation of Ngn3 expression. Third, data from in vitro and in vivo models revealed that ZEB1/Ngn3 signaling is critical for CSC pool expansion by enhancing the frequency of the SD population. Finally, we show that the expression of ZEB1 is increased in samples from breast cancer patients and is inversely correlated with NGN3 protein levels. Therefore, our results indicated that ZEB1/Ngn3 signaling may determine the replenishment of the breast CSC pool and promote tumor initiation. Although ZEB1 is a well-characterized EMT regulator, several pieces of evidence suggest that it also contributes to the acquisition of CSC properties via a combination of genetic, epigenetic, and transcriptional mechanisms [31]. For example, ZEB1 interacts with HDAC components at the E-cadherin promoter in either a miR-200-dependent [32] or miR-200-independent manner [27], thus leading to induction of a mesenchymal-like phenotype and to increased tumorigenic potential. These findings suggest that ZEB1 might control tumor stemness by influencing the EMT process. However, Zhang et al. have reported that ZEB1 affects CSC-associated properties, such as radioresistance, independently of its ability to induce the EMT program, thus suggesting that adoption of a mesenchymal phenotype is not necessarily related to tumor onset [20]. In the present study, an essential role for ZEB1 in tumor initiation and maintenance in vitro and in vivo was confirmed by deleting ZEB1 in pre-existing breast cancer cells (MDA-MB-231 and SUM-159). Together, these findings demonstrate that ZEB1 may exert different tumorigenic functions (i.e., Figure 6: ZEB1 and NGN3 protein expression levels are inversely correlated in human breast cancer. (A) Representative images of immunohistochemical staining of ZEB1, NGN3, and ALDH1 in serial sections of the same tumor from three cases. Scale bars, 50 μm. (B) The expression score for NGN3 indicates a negative correlation with ZEB1 expression in the 156 human breast cancer samples analyzed. (C) The expression score for ALDH1 indicates a positive correlation with ZEB1 expression. (D) The expression score for ALDH1 indicates a negative correlation with NGN3 expression. (E) The expression of ZEB1 is positively correlated with the histological grades of breast cancer. (F) The expression of NGN3 is negatively correlated with the histological grades of breast cancer. All data were analyzed by Spearman’s rank correction test. www.impactjournals.com/oncotarget 54396 Oncotarget stemness maintenance and EMT) that are not necessarily interrelated. Past studies of ZEB1 transcriptional activity were mostly focused on its impact within the EMT gene network, such as downregulation of epithelial cell markers (E-cadherin, PATJ, Crumbs3) and of some miR-200 family members that control epithelialmesenchymal specification, and upregulation of mesenchymal markers (v.g. vimentin, N-cadherin, and matrix metalloproteinases [33]. On the other hand, ZEB1 has been shown to affect stemness features indirectly, through transcriptional repression of miRNAs such as miR-200 and miR-203 [18]. Here, we identified Ngn3 as a direct ZEB1 target that mediates its effects on CSC maintenance and replenishment. NGN3 is a basic helixloop-helix transcription factor required for controlling cell fate differentiation in multipotent progenitor cells during embryogenesis. For example, during steady state spermatogenesis, Ngn3 expression marks a transition from strong spermatogonial stem cell potential to committed transit-amplifying cells that normally proliferate and differentiate [34]. Ngn3 also acts as a genetic switch in pluripotent pancreatic progenitor cells, by specifying an endocrine cell fate [35]. The importance of Ngn3 in the control of endocrine cell differentiation can also be observed in the stomach and intestine [36]. Notably, Lee et al. have reported that Ngn3-/- mice display intestinal metaplasia (IM) of the gastric epithelium [37]. Given that IM is an important risk factor for gastric cancer and an inevitable stage in gastrocarcinogenesis [38], these observations suggest an inhibitory effect of Ngn3 on the appearance of CSC characteristics that is in agreement with our data from in vitro and in vivo breast cancer models (Supplementary Figure 10 and 11). Thus, downregulation of Ngn3 expression is at least partially involved in the mechanism by which ZEB1 promotes breast cancer cell stemness and tumor initiation and progression. Although mainly recognized by its silencing effects on epithelial gene expression, ZEB1 can also mediate direct transcriptional activation of target genes, depending on the nature of the co-factors recruited [39, 40]. Gene silencing has been shown to occur during ZEB1 complexation with PC2-CtBP-LSD1-LCoR, or with the chromatin-remodeling protein BRG [41]; recruitment of Smad3-p300-P/CAF, in contrast, results in transcriptional activation [42]. Here we provide an alternative mechanism for ZEB1/Ngn3–mediated stemness acquisition in breast cancer cells that involves methylation and deacetylation of the Ngn3 promoter. Namely, upon ZEB1 interaction with DNMT3B and HDAC1, the ensuing promoter hypermethylation and histone deacetylation result in repression of Ngn3 expression, an effect readily reversed by inhibiting DNA methylation with AZA and histone deacetylation with TSA. Accordingly, by examining 156 primary breast cancer specimens, we confirmed that ZEB1 is overexpressed in cancer tissues, and its www.impactjournals.com/oncotarget expression is inversely correlated with that of NGN3. Thus, dysregulation of Ngn3 may provide a mechanistic link between the ectopic expression of ZEB1 and the ontogeny of aggressive breast cancer. These results were further supported by our analysis of cell division variants (symmetric versus asymmetric) in CSCs, which evidenced the critical role of ZEB1/Ngn3 signaling in replenishing the breast CSC pool and promoting tumorigenesis. Similarly to our own conclusions regarding ZEB1, the polarity of cell division has been shown to be affected by p53 in murine mammary stem cells; in this model, p53 loss leads to uniform NUMB redistribution in the stem cell undergoing mitosis, enhances mammospheres’ replication potential, and increases the rate of SD [43]. Although in such study the underlying mechanism remained undefined, a recent study implicated ZEB1 as a direct transcriptional target of p53 [44]; thus we believe that ZEB1 signaling may be involved. In summary, our study uncovered a key role for ZEB1 in the establishment of CSC properties and in promoting tumor initiation by breast CSCs. Since ZEB1 may affect different steps in the tumorigenic process, more detailed studies are needed to elucidate its possible contribution to tumor initiation and expansion. In addition, given that ZEB1 downregulation parallels metastatic colonization [45, 46], its possible contribution to the metastatic process should also be addressed. MATERIALS AND METHODS Cell culture and transfection Human breast cancer cell lines were maintained in DMEM (SUM-159) and RPMI 1640 (MDA-MB-231) supplemented with 10% FBS, 100 IU penicillin, and 100 mg/mL streptomycin. Cells were transfected using Lipofectamine 2000 (Invitrogen, California, USA) according to the manufacturer’s protocol. Plasmid construction The human cDNA fragment encoding full-length ZEB1 and Ngn3 was prepared by PCR and cloned into pLV-EF1-MCS-IRES-Bsd (Biosettia, San Diego, USA). The lentiviral-based vector pLV-H1-EF1α-puro (Biosettia, San Diego, USA) was used to express shRNAs in breast cancer cells. The human Ngn3 full-length and truncated promoter sequences were obtained by PCR from human genomic DNA and cloned into the pGL3-basic vector (Promega, Wisconsin, USA). Primer sequences are listed in Supplementary Data. Generation of lentiviruses Lentiviruses were generated by transfecting subconfluent HEK293T cells with lentiviral vectors and 54397 Oncotarget packaging plasmids via calcium phosphate transfection. Viral supernatants were collected 48 h after transfection, resuspended, and filtered through 0.45 μm filters (Millipore, Massachusetts, USA). MDA-MB-231 or SUM-159 cells were transduced with lentivirus-containing medium and selected with antibiotic. After one week, cell pools were obtained and expanded. absence of the ALDH inhibitor diethylaminobenzaldehyde. To identify the CD44+CD24- population, CD44-APC and CD24-PE primary antibodies (BD, USA) were incubated with single cells in PBS containing 2% FBS for 30 minutes at 4°C. Then, cells were washed twice with PBS, resuspended in 1 mL PBS, and kept on ice until FACS analysis. To identify the side population (SP) and nonside population fractions, cells were trypsinized and suspended at a concentration of 1x106 cells/mL in PBS containing 2% FBS. Hoechst 33342 dye (Sigma-Aldrich, Vienna, Austria) was added at a final concentration of 5 μg/mL in the presence or absence of 100 μM verapamil (Sigma-Aldrich), and the cells were incubated at 37°C for 90 min with intermittent shaking every 15 min. The cells were washed twice with PBS and resuspended in 1 mL PBS with 1 μg/mL propidium iodide and kept on ice until FACS analysis. Cell analyses were carried out with a FACSAria instrument (BD, USA). RNA extraction and quantitative RT-PCR Total RNA (0.5 μg) from each sample was collected using TRIzol reagent (Invitrogen, California, USA), and first-strand cDNA synthesis was performed using M-MLV Reverse Transcriptase (Takara, Tokyo, Japan). The specific products were amplified by quantitative PCR using a TransStart Green Q-PCR SuperMix Kit (TransGen, Beijing, China). GAPDH was used as a normalization control. Primer sequences are listed in Supplementary Data. Methylation assays Luciferase assay DNA was extracted from breast cancer cells and processed for bisulfite treatment (Qiagen, Hilden, Germany). Bisulfite-treated DNA was then used to examine the methylation status of the CpG islands in the Ngn3 promoter, using Bisulfite-Sequencing PCR (BSP) according to the manufacturer’s protocol (Roche, Basel, Switzerland). Primer sequences are listed in Supplementary Data. Cells were co-transfected with full-length or truncated human Ngn3 promoters and ZEB1 expression plasmid in 24-well plates. Lysates were prepared 36 h after transfection, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Wisconsin, USA) according to the manufacturer’s protocol. Luciferase activity was normalized to Renilla luciferase activity. Immunoprecipitation assay Immunoblotting assay Cell lysates were incubated with specific antibodies and Protein G agarose beads (Invitrogen, California, USA) at 4°C overnight, then washed three times with a buffer containing 50 mM Tris (pH 7.5), 100 mM NaCl, 7.5 mM EGTA, and 0.1% Triton X-100. The antibodies used for immunoprecipitation are shown in Supplementary Data. The preparation of total cell extracts and immunoblotting with appropriate antibodies (indicated in Supplementary Data) were performed as previously described [47]. Labeled proteins were visualized with an ECL chemiluminescence kit (Millipore, Massachusetts, USA). Chromatin immunoprecipitation Tumorsphere assay ChIP assays were performed using an EZ-ChIP kit (Millipore, Massachusetts, USA) according to the manufacturer’s instructions. The antibodies used in these experiments are shown in Supplementary Data. The fragments of human Ngn3 promoter containing the E2-box 1, E2-box 2 and E2-box 3 elements in immunoprecipitates were amplified by quantitative PCR. Primer sequences are listed in Supplementary Data. Single-cell suspensions were prepared in sphereculturing medium (Stemcell Technologies) and seeded in 24-well ultra-low attachment plates (Corning) at 1,000 cells/well. The number of wells with spheres was recorded on day 14 after culturing. Tumorsphere formation was monitored using an inverted Leica microscope fitted with a camera. Sphere numbers and diameters were imaged and quantified in >5 fields per sample. Tissue samples Flow cytometry A total of 156 human breast cancer specimens were obtained from the General Hospital of the People’s Liberation Army (PLAGH, Beijing, China). All patients had histologically confirmed invasive ductal carcinoma of the breast and were recruited by the same department. This The ALDEFLUOR assay (Stemcell Technologies) was performed according to the manufacturer’s guidelines to identify cells with high ALDH activity. Cells were incubated for 40 minutes at 37 °C in the presence or www.impactjournals.com/oncotarget 54398 Oncotarget study was approved by the institutional ethics committees at PLAGH and the Medical College of Nankai University. incubated for 45 minutes in a shaker (37°C, 90 rpm). The dissociated cells were filtered through 40 μm cell strainers (BD, USA) and concentrated by centrifugation at 1,500 rpm for 15 minutes at RT. Cells were washed with 50 mL volumes of PBS until the supernatant was clear of red blood cells. Immunohistochemical analysis Immunohistochemical analysis of paraffinembedded sections was performed using an Envision Kit (Dako, Denmark) according to the manufacturer’s protocol. Sections were boiled in retrieval solution to expose antigens. Specific antibodies (see Supplementary Data) were applied to the sections. Slides were counterstained with hematoxylin, dehydrated, and mounted. Immunostaining was independently evaluated by 2 pathologists. Statistical analysis Statistical analyses were performed using SPSS 13.0 software (SPSS, Chicago, IL, USA). Data are presented as the means ± SD and represent three independent experiments. Spearman’s rank correlation test was used to analyze the correlation of gene expression in tissue samples. One-way analysis of variance (ANOVA) was used to compare means between treatment groups. Where appropriate, Student’s t-test for unpaired observations was applied. A P-value < 0.05 was considered significant. Cell division assay CD44+CD24- cells were treated with 20 mM blebbistatin (Selleck Chemicals, USA) for 48 hours in suspension culture and fixed for 20 minutes in 10% neutral buffered formalin at -20°C. Fixed cells were blocked for 1.5 hours with 5% goat serum and then incubated with anti-Numb and anti-CD44 primary antibodies diluted in TBST (containing 0.1% Tween 20 and 5% BSA) overnight at 4°C. Then, cells were washed 3 times with TBST at RT, incubated with secondary antibodies for 1 hour, then washed 3 times at RT. The secondary antibodies were DyLight 488 goat anti-mouse and DyLight 594 goat antirabbit. Cell nuclei were counterstained and mounted with Prolong Gold Antifade Reagent with DAPI (Molecular Probes) overnight at RT. ACKNOWLEDGMENTS This work was supported by grants from the International S&T Cooperation Program of China (No. 2015DFA30420), and the National Natural Science Foundation of China (No. 81472545; No. 31428013; No. 81372386). CONFLICTS OF INTEREST The authors have no conflicts of interest. REFERENCES Tumor xenograft experiments 1. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001; 414:105–11. All experimental procedures involving animals were performed according to the institutional ethical guidelines for animal experiments and approved by the Ethics Committee for Animal Use at the Medical College of Nankai University. Briefly, cells were collected and suspended at different concentrations (1.0×107, 5.0×106, 2.5×106, 1.25×106, and 6.25×105 cells/mL) in 200 μL PBS, and then injected into the mammary fat pads of female BALB/c nude mice. After 15 days, mice were euthanized, and the tumor tissues were processed for detection of the CD44+CD24- population and ALDH activity and sectioned for histological evaluation. 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W3046552576.txt

OsFD4 promotes the rice floral transition via florigen activation complex formation in the shoot apical meristem Martina Cerise, Francesca Giaume, Mary Galli, Bahman Khahani, Jérémy Lucas, Federico Podico, Elahe Tavakol, François Parcy, Andrea Gallavotti, Vittoria Brambilla, et al. To cite this version: Martina Cerise, Francesca Giaume, Mary Galli, Bahman Khahani, Jérémy Lucas, et al.. OsFD4 promotes the rice floral transition via florigen activation complex formation in the shoot apical meristem. New Phytologist, 2021, 229 (1), pp.429-443. �10.1111/nph.16834�. �hal-02953028� HAL Id: hal-02953028 https://hal.science/hal-02953028 Submitted on 1 Oct 2020 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. 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Distributed under a Creative Commons Attribution 4.0 International License 1 OsFD4 promotes the rice floral transition via Florigen Activation Complex formation in the 2 shoot apical meristem 3 4 Martina Cerise1,6, Francesca Giaume1, Mary Galli3, Bahman Khahani4, Jérémy Lucas5, Federico 5 Podico1, Elahe Tavakol4, François Parcy5, Andrea Gallavotti3, Vittoria Brambilla2 and Fabio 6 Fornara1,* 7 8 1 Department of Biosciences, University of Milan, Milan, 20123 Italy 9 2 Department of Agricultural and Environmental Sciences, University of Milan, Milan, 20123 Italy 10 3 Waksman Institute of Microbiology, Rutgers University, Piscataway, New Jersey, NJ 08854, 11 United States 12 4 13 Shiraz, 71441-65186, Iran 14 5 15 38054, Grenoble, France 16 6 17 Cologne, D-50829, Germany Department of Crop Production and Plant Breeding, College of Agriculture, Shiraz University, University Grenoble Alpes, CNRS, CEA, INRAE, IRIG-DBSCI-LPCV, 17 avenue des martyrs, F- Max Planck Institute for Plant Breeding Research, Department of Developmental Biology, 18 19 * Correspondence: Fabio Fornara (fabio.fornara@unimi.it), Tel. +390250314817 20 21 Total word count: 6515 22 Introduction word count: 1163 23 Materials and Methods word count: 852 24 Results word count: 2628 25 Discussion word count: 1866 26 Number of figures: 6 27 28 Heading title: OsFD4 promotes flowering in rice 29 30 Associated twitter accounts: @fornaralab, @vittoriabr, @Francois_Parcy 31 32 33 34 1 35 ORCID DETAILS 36 Martina Cerise, https://orcid.org/0000-0002-9654-252X 37 Francesca Giaume, https://orcid.org/0000-0002-6074-6950 38 Bahman Khahani, https://orcid.org/0000-0003-4499-2825 39 Elahe Tavakol, https://orcid.org/0000-0001-9033-7237 40 François Parcy, https://orcid.org/0000-0003-2191-500X 41 Andrea Gallavotti, https://orcid.org/0000-0002-1901-2971 42 Vittoria Brambilla, https://orcid.org/0000-0003-0673-6898 43 Fabio Fornara, https://orcid.org/0000-0002-1050-0477 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 2 69 70 SUMMARY • In rice, the florigens Heading Date 3a (Hd3a) and Rice Flowering Locus T 1 (RFT1), OsFD- 71 like 72 Activation/Repressor Complexes (FACs/FRCs), which regulate transition to flowering in 73 leaves and apical meristem. Only OsFD1 has been described as part of complexes promoting 74 flowering at the meristem, and little is known about the role of other bZIP transcription 75 factors, the combinatorial complexity of FAC formation, and their DNA binding properties. 76 • bZIP transcription factors, and Gf14 proteins assemble into Florigen Here, we used mutant analysis, protein-protein interaction assays and DAP-sequencing 77 coupled to in silico prediction of binding syntaxes, to study several bZIP proteins that 78 assemble into FACs or FRCs. 79 • We identified OsFD4 as component of a FAC promoting flowering at the shoot apical 80 meristem, downstream of OsFD1. The osfd4 mutants are late flowering and delay expression 81 of genes promoting inflorescence development. Protein–protein interactions indicate an 82 extensive network of contacts between several bZIPs and Gf14 proteins. Finally, we identified 83 genomic regions bound by bZIPs with promotive and repressive effects on flowering. 84 • We conclude that distinct bZIPs orchestrate floral induction at the meristem and that FAC 85 formation is largely combinatorial. While binding to the same consensus motif, their DNA 86 binding syntax is different, suggesting discriminatory functions. 87 88 89 90 Key words: bZIP, florigen activation complex, photoperiodic flowering, rice, shoot apical meristem. 91 92 93 94 95 96 97 98 99 100 101 102 3 103 INTRODUCTION 104 When external and internal conditions are favourable, plants switch their life cycle from the 105 vegetative to the reproductive phase. This process is called floral transition and is determined by 106 events taking place in both leaves and shoot apical meristem (SAM). In rice (Oryza sativa), transition 107 to the reproductive phase is controlled by changes in the photoperiod and is accelerated when day 108 length falls under a critical threshold. Because of this response, rice is classified as short day (SD) 109 plant. Yet, it can flower also under non-inductive long day (LD) conditions, after a prolonged 110 vegetative phase. As such, its photoperiodic response is facultative. 111 The molecular events that guide the floral transition start in the leaves when transcription of Heading 112 date 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1) takes place in response to inductive 113 photoperiods. Hd3a and RFT1 are homologs of Arabidopsis FLOWERING LOCUS T (FT) and 114 members of the phosphatidyl ethanolamine-binding protein (PEBP) family (Kojima et al., 2002; 115 Komiya et al., 2008, 2009). Similarly to FT, they are transcribed in companion cells of leaves; 116 subsequently, their cognate proteins can move through the phloem to reach the SAM, where they 117 promote expression of genes necessary to activate the inflorescence development program (Tamaki 118 et al., 2007; Komiya et al., 2009). Analysis of RNA interference lines and CRISPR mutants 119 demonstrated that a delay in flowering occurs upon downregulation of Hd3a or RFT1 (Komiya et al., 120 2008, Liu et al., 2019). However, the phenotypic effects of reduced expression of Hd3a or RFT1 121 depend on the photoperiod. Under SD, Hd3a RNAi lines delay flowering and double RFT1-Hd3a 122 RNAi plants completely block it, indicating that both florigens can redundantly promote the floral 123 transition (Komiya et al., 2008; Tamaki et al., 2015). Conversely, RFT1 RNAi lines delay flowering 124 under LD only, indicating that RFT1 can promote flowering also under non-inductive photoperiods 125 (Komiya et al., 2009). This difference correlates with the expression of Hd3a and RFT1 in leaves: 126 when plants grow under SD conditions, both florigens are transcribed, whereas growth under non- 127 inductive LD conditions promotes RFT1 expression only (Komiya et al., 2008, 2009). Differential 128 sensitivity of Hd3a and RFT1 transcription to day length is partly explained by distinct promoter 129 architectures (Komiya et al., 2008, 2009). 130 Several positive and negative regulators control expression of the florigens in leaves. Among them, 131 the zinc finger transcription factor Heading date 1 (Hd1) , homolog of Arabidopsis CONSTANS, 132 promotes expression of Hd3a and RFT1 under SD, while it represses it under LD (Yano et al., 2000; 133 Hayama et al., 2003). Hd1 functions as transcriptional regulator through physical interaction with 134 Grain yield, plant height and heading date 8 (Ghd8), Ghd7 and OsNF-YC7. Ghd8 and OsNF-YC7 135 are the B and C subunits of a heterotrimeric NF-Y complex, respectively, that assemble with Hd1, 136 bind the promoter of Hd3a and repress its transcription (Goretti et al., 2017; Zhu et al., 2017; Du et 4 137 al., 2017). Ghd7 encodes for a CCT domain protein that represses flowering (Xue et al., 2008; 138 Nemoto et al., 2016) . 139 The B-type response regulator Early Heading date 1 (Ehd1) is a major flowering promotor that shares 140 no homology with other genes of Arabidopsis thaliana (Doi et al., 2004). Ehd1 is expressed at higher 141 levels under SD conditions and preferentially promotes Hd3a transcription (Zhao et al., 2015). In 142 agreement with its role as a flowering activator, ehd1 mutants show a late flowering phenotype 143 compared to wild type, whereas Ehd1 overexpression triggers early flowering under both SD and LD 144 conditions (Doi et al., 2004; Itoh et al., 2010; Cho et al., 2016). Hd1 can repress Ehd1 mRNA 145 expression under LD, by directly binding to its promoter (Nemoto et al., 2016). These genetic 146 interactions are at the core of the photoperiodic flowering pathway in rice, which exhibits a different 147 architecture compared to the one of Arabidopsis (Andrés & Coupland, 2012). 148 When florigenic proteins are produced, they influence downstream gene expression by assembling 149 into Florigen Activation or Repressor Complexes (FACs/FRCs), higher-order complexes containing 150 PEBP molecules and FD-like bZIP transcription factors (Tsuji et al., 2013; Brambilla et al., 2017; 151 Kaneko-Suzuki et al., 2018). The latter allow the complex to bind DNA at consensus sites 152 characterized by the core sequence ACGT (Izawa et al., 1993). Interactions between florigens and 153 bZIPs can occur either directly or indirectly via bridging proteins called Gf14s, members of the 14- 154 3-3 protein family (Taoka et al., 2011). In leaves, FACs/FRCs regulate expression of the florigens, 155 generating positive and negative feedback loops controlling Ehd1 expression (Brambilla et al., 2017). 156 More specifically, FRCs composed by the florigens and the bZIP transcription factor Hd3a BINDING 157 REPRESSOR FACTOR 1 (HBF1) delay the floral transition, as rice loss-of-function HBF1 mutants 158 flower earlier compared to the wild type. HBF1 can bind the promoter of Ehd1 to reduce its expression 159 in leaves, thus generating a negative feedback loop on Hd3a and RFT1 production, that likely 160 modulates the amount of florigens being produced (Brambilla et al., 2017). 161 Another bZIP transcription factor, OsFD1, encodes an interactor of the florigens and is part of a 162 flowering-promoting FAC (Taoka et al., 2011). OsFD1 is expressed in both leaves and SAM and 163 interacts with Hd3a and RFT1 only indirectly via Gf14 proteins. In leaves, OsFD1 promotes the 164 expression of Ehd1 generating a positive feedback loop on florigen production, whereas in the SAM, 165 OsFD1-containing FACs promote transcription of OsMADS14 and OsMADS15, two key activators 166 of flower development in rice (Taoka et al., 2011; Kobayashi et al., 2012; Zhao et al., 2015; Wu et 167 al., 2017). Thus, the florigens not only connect leaves and SAM to convey seasonal information to 168 the apex, but their movement throughout the plant allows formation of distinct florigen-containing 169 complexes in different tissues and with diversified functions. 5 170 In the SAM, OsFD1 can further switch from activating to repressing flowering by forming FRCs. In 171 FRCs, 172 CENTRORADIALIS 1 (RCN1), RCN2, RCN3 and RCN4 which are PEBP and members of the TFL1- 173 like subclade (Kaneko-Suzuki et al., 2018). They are expressed in the rice vascular tissue and, 174 similarly to Hd3a and RFT1, can move through the phloem and reach the SAM where they bind 175 OsFD1 via Gf14s, to regulate production of secondary branches of the panicle (Kaneko-Suzuki et al., 176 2018). 177 Despite its central position in molecular complexes controlling flowering, osfd1 RNA interference 178 plants showed almost no flowering delay compared to wild type plants (Taoka et al., 2011). Its 179 interaction in both FACs and FRCs with opposite effects on flowering could partially explain such 180 phenotype. However, it cannot be excluded that additional bZIP transcription factors could operate 181 in parallel or redundantly with OsFD1. 182 Here, we functionally characterize OsFD4, an FD-like bZIP transcription factor promoting the floral 183 transition under both inductive and non-inductive photoperiodic conditions. We determine the extent 184 of OsFD4 interactions in FACs and its genetic relationship with OsFD1. Using Positional Weight 185 Matrices (PWM) gathered from DNA Affinity Purification sequencing (DAP-seq), we interrogate the 186 binding of OsFD1, OsDF4 and HBF1 to promoters of target genes, starting to define their distinct 187 modes of DNA binding. OsFD1 interacts with four different CENTRORADIALIS-like proteins, RICE 188 189 MATERIALS AND METHODS 190 Plant growth conditions, RNA preparation and quantification of gene expression 191 The Nipponbare and Dongjin ecotypes (Oryza sativa L.) were used in this study. Plants were grown 192 in phytotrons under long-day conditions (14,5h light/9,5h dark) or short-day conditions (10h light/14h 193 dark). For quantification of gene expression using qRT-PCR, SAM samples were collected at 194 Zeitgeber (ZT) 0 in time courses of several days. Three biological replicates were performed for 195 osfd4-1 time-courses. Two biological replicates were sampled for osfd1-1 time courses. SAMs were 196 manually dissected under a stereomicroscope and the samples included the meristem proper as well 197 as some small meristematic leaves. For leaf samples, the distal part of mature leaves was used. RNA 198 was extracted with TRIzol® (Termofisher Scientific) following manufacturer’s instructions. The 199 cDNA was retrotranscribed with Im-Prom-II RT (Promega) using a polyT primer and 1 µg of total 200 RNA as starting template. Quantification of transcripts was performed with the primers listed in Table 201 S1 using a RealPlex2 thermocycler. The Maxima SYBR qPCR master mix (Termofisher Scientific) 202 was used in qRT-PCR experiments. Three technical replicates were performed for each qRT-PCR. 203 6 204 Protein-protein interaction assays 205 Yeast two hybrid was performed cloning the coding sequences into the vectors pGADT7 and 206 pGBKT7 (Clontech) and transformed into AH109 and Y187 yeast strains, respectively. Interactions 207 were tested by mating and growth on selective drop out media without the aminoacid -L-W-H-A 208 according to Brambilla et al., 2017. 209 BiFC experiments were performed using the vectors pBAT TL-B sYFP-N and pBAT TL-B sYFP-C. 210 Clones were transformed in tobacco epidermal cells by Agro-infiltration. Infiltrated leaves were 211 observed at the confocal 24 h after infiltration. Each interaction was tested in three different replicates. 212 213 CRISPR-Cas9 cloning and Rice transformation 214 The CRISPR-Cas9 vectors previously described by Miao et al., 2013 were used. We designed one 215 single-guide RNA for OsFD1, OsFD4 and OsFD3 (Table S1). We obtained edited osfd4, osfd3 and 216 osfd1 mutants starting from Nipponbare calli produced according to the protocol of Sahoo et al., 2011. 217 Rice calli were transformed using EHA105 Agrobacterium tumefaciens strain. Transformed calli 218 were selected on two selection media containing 50 mg/L and 100 mg/L of hygromycin, respectively. 219 After regeneration, we genotyped transformant plants via by sequencing PCR products amplified 220 across the single-guide RNA targeted region. 221 222 DAP seq assay 223 pENTR clones containing the OsBZIP42, OsBZIP69, and OsBZIP77 ORFs were recombined into the 224 pIX-HALO vector and processed according to Bartlett et al., 2017. We used 1 ug of pIX-HALO-TF 225 plasmid DNA for protein expression in the TNT rabbit reticulocyte expression system (Promega). 226 For DNA binding reactions, 500 ng of adapter-ligated library prepared from genomic DNA extracted 227 from Nipponbare leaves was used. Libraries were sequenced on a NextSeq500 with 75bp single end 228 reads. A GST-HALO negative control sample was also processed in parallel and used for background 229 subtraction in peak calling. 230 231 DAP-seq analysis 232 Raw reads were trimmed using Trimmomatic (Bolger et al., 2014) with the following parameters: 233 ILLUMINACLIP:TruSeq3-SE:2:30:10 234 MINLEN:50. Trimmed reads were mapped to the Osativa v7 reference genome (release 40; 235 https://plants.ensembl.org/Oryza_sativa/) using Bowtie2 v2.2.8 (Langmead & Salzberg, 2012) with 236 default parameters. Mapped reads were filtered for reads aligning only to a single location in the 237 genome. Peaks were called using the GEM peak caller (Guo et al., 2012) as described in Galli et al., LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 7 238 2018. PAVIS (Huang et al. 2013) was used to identify putative target genes. Target genes were 239 assigned based on the gene located closest to each peak and were restricted to those peaks located 240 5kb upstream of the TSS, 3kb downstream of the TES, or located within the gene. The 2000 interval 241 relative to the TSS was surveyed for detecting the position of peaks and distribution of them using a 242 bin size of 25 bp. 243 244 Comparison between targets of AtFD, OsFD1, OsFD4 and OsHBF1 245 In order to compare the FD-bound regions in Arabidopsis and rice, we retrieved ChIP-seq data for 246 FD from Collani et al. (2019). We next identified putative rice orthologs using the EnsemblPlants 247 (https://plants.ensembl.org/index.html) database (Table S2). The overlap between corresponding 248 genes bound by AtFD, OsFD1, OsFD4 and HBF1 was illustrated using a Venn diagram (Oliveros, 249 2007). 250 251 Motif enrichment analysis and spacing/syntax analysis 252 Peaks identified by the GEM (Guo et al., 2012) peak caller were used to compute motif enrichment, 253 using the MEME-suite (default arguments). Generated motifs were evaluated using ROC analysis to 254 estimate their prediction power, i.e. their capability to differentiate between bound and not bound 255 regions. For this, a negative set of non-bound regions with similar features (same length, same %GC, 256 same genomic origin [exonic, promoter,...] as the bound sequences) was generated as previously 257 described (Stigliani et al., 2019). Detection of preferred spacings between motifs was done using the 258 called peaks and the MEME-generated position weight matrix (PWM). Predicted individual binding 259 sites were detected for a given threshold, for sets of both bound and unbound regions. Spacing 260 between predicted binding sites were computed for both sets. Enrichment was calculated by 261 comparing bound and not bound sets of regions. The PWM threshold values were empirically chosen: 262 we used a range of threshold values to ensure robustness of the result. Detailed methods are available 263 in Stigliani et al., 2019. 264 265 RESULTS 266 OsFD4 promotes flowering in rice 267 In leaves, HBF1- and OsFD1-containing complexes control production of the florigens by regulating 268 Ehd1 expression (Brambilla et al., 2017). In the SAM, the OsFD1-containing FAC is known to 269 promote the floral transition by directly inducing expression of OsMADS14 and OsMADS15 (Taoka 270 et al., 2011). Yet, OsFD1 RNA interference plants showed only a marginally significant flowering 271 delay compared to the wild type, in sharp contrast to the phenotype shown by Hd3a-RNAi, RFT18 272 RNAi and Hd3a/RFT1 double RNAi plants (Komiya et al., 2008). This suggests that other bZIP 273 transcription factors might share similar functions and compensate for reduced OsFD1 expression. 274 We previously explored the bZIP family, searching for members having positive or negative effects 275 on the floral transition (Brambilla et al., 2017). Here, we describe the functional characterization of 276 bZIP69/OsFD4 (LOC_Os08g43600). 277 After searching the PFG rice T-DNA collection we identified line 2D_41663 in the Dongjin variety 278 that harboured a T-DNA insertion in the 3’-UTR of OsFD4 (Fig. S1). The mutant line was renamed 279 osfd4-1. We could not detect OsFD4 mRNA expression in osfd4-1 (Fig. S1). Heading dates of the 280 mutant plants were assessed under both SD and LD, alongside Dongjin wild type controls. Mutant 281 plants flowered late under both conditions (Fig. 1a). 282 To validate the osfd4-1 mutant phenotype, we generated additional osfd4 mutant alleles using 283 CRISPR-Cas9 site-directed mutagenesis in the Nipponbare background (Miao et al., 2013). We also 284 generated novel osfd1 mutants to compare single mutant phenotypes. Finally, since we previously 285 showed that OsFD4 strongly interacts with bZIP24/OsFD3 (Brambilla et al., 2017), we also generated 286 osfd3 mutants (Fig. S1). Homozygous plants of the T1 generation were grown for two months under 287 non-inductive LD conditions and then shifted to inductive SD to score heading dates. As shown in 288 Fig. 1b and Fig. S1, osfd4-3 showed delayed flowering compared to the wild type, confirming the 289 osfd4-1 phenotype. The osfd1-1 mutant flowered later than both wild type and osfd4-3, showing a 290 strong delay (Fig. 1b-d). The difference compared to published OsFD1-RNAi mutants could be due 291 to the different conditions used between studies, to the different genetic backgrounds used, or to a 292 limited reduction of OsFD1 mRNA in RNAi lines. No effect on heading could be observed in osfd3- 293 1 mutants (Fig. 1b). 294 To investigate the possible functional redundancy between OsFD4 and OsFD1, we crossed osfd4-1 295 and osfd1-2 mutants to generate an osfd4-1 osfd1-2 double mutant (note that the genetic background 296 is mixed between Nipponbare and Dongjin). Flowering time experiments under inductive conditions 297 demonstrated that no additive effect could be detected in the osfd4-1 osfd1-2 double mutant relative 298 to osfd1-2 (Fig. 1d), i.e. the phenotypic effect observed was that of osfd1-2 single (Fig. 1b-d). These 299 data indicate that OsFD4 encodes a promotor of flowering in rice and that the effect of osfd4 and 300 osfd1 mutants is not additive. 301 302 OsFD4 is expressed in the shoot apical meristem and is regulated by OsFD1 303 OsFD1 is expressed in both leaves and SAM. It promotes flowering, having distinct functions in both 304 tissues during the floral transition (Taoka et al., 2011; Tamaki et al., 2015; Jang et al., 2017; 305 Brambilla et al., 2017). We quantified the expression of OsFD4 mRNA in leaves and apical 9 306 meristems, using qRT-PCR. Transcripts of OsFD4 were detected in apical meristems but not in leaves 307 during the transition to flowering (Fig. 2a,b). Quantifications showed that OsFD1 and OsFD3 308 expression was higher relative to OsFD4 expression in both tissues. 309 To verify the epistatic interaction between OsFD1 and OsFD4, observed in the osfd4-1 osfd1-2 310 double mutant, we analysed OsFD4 and OsFD1 mRNA levels in osfd1-1 and osfd4-1 mutants, 311 respectively. We observed that OsFD4 expression was decreased in osfd1-1 compared to the wild 312 type (Fig. 2c), whereas OsFD1 expression was unchanged in osfd4-1 mutants compared to Dj wild 313 type (Fig. 2d). These data indicate that OsFD1 is an upstream promotor of OsFD4 expression, in 314 agreement with the phenotype of double osfd4-1 osfd1-2 mutants (Fig. 1d). 315 316 Network of interactions among FAC components 317 The rice florigens interact with bZIP transcription factors either directly or through Gf14 proteins. 318 Interactions are likely dynamic and combinatorial, giving rise to several complexes, some also with 319 repressive functions (Zhao et al., 2015; Jang et al., 2017; Brambilla et al., 2017; Kaneko-Suzuki et 320 al., 2018). Dimers between Gf14 proteins form a W-shaped scaffold that bridges interactions with 321 bZIPs and with the florigens. We found that extensive combinatorial interactions are possible between 322 Gf14s expressed at the apex, giving rise to homo- and heterodimers (Fig. S2, all mating controls are 323 shown in Fig. S3). Interactions between Gf14s and bZIPs occur via the C-terminal SAP-domain, 324 which is common among several bZIPs, including OsFD4 and OsFD3 (Fig. S1). We tested whether 325 OsFD4 could interact with components of FACs. We first tested the binding between OsFD4 and the 326 six Gf14s expressed in the SAM, including Gf14A to Gf14F (Purwestri et al., 2009). All six Gf14 327 proteins interacted with OsFD4 in Yeat-2-Hybrid (Y2H), but the strongest interactions were detected 328 between OsFD4 and Gf14A and Gf14F (Fig. 3a). Weaker yeast growth on -L-W-H-A selective media 329 was observed with other OsFD4-Gf14 combinations. Note that in a previous report we did not detect 330 the OsFD4-Gf14C interaction, using -L-W-H +3AT media (Brambilla et al., 2017), consistent with 331 the findings of Kim et al., 2016. Thus, interactions between OsFD4 and Gf14B, C, D, and E are likely 332 weak. Also, OsFD3 could interact with all Gf14s tested (Fig. S2). 333 We next asked if all Gf14 proteins expressed in the SAM could interact with RFT1 and Hd3a. Both 334 florigens interacted with all the Gf14s tested, suggesting that in the SAM, OsFD4, OsFD3 and OsFD1 335 can potentially form FACs indirectly via all 14-3-3 proteins (Fig. 3b). To confirm Y2H findings, we 336 performed BiFC with OsFD4 and Gf14B and observed reconstituted YFP expression in nuclei 337 indicating a positive interaction (Fig. S2). 338 Since HBF1 and HBF2 can directly contact Hd3a without the bridging function of Gf14s (Brambilla 339 et al., 2017), we tested direct interactions between OsFD4 and Hd3a or RFT1. Using Y2H, we found 10 340 that OsFD4 can directly associate with RFT1, but no interaction was detected between OsFD4 and 341 Hd3a, whereas OsFD3 could interact with both florigens (Fig. 3c). These data are consistent with a 342 recent independent study, indicating that OsFD4 preferentially binds to one florigen only (Jang et al., 343 2017). We then used BiFC to further test the OsFD4-RFT1 interaction, as well as to determine its 344 subcellular localization. We detected fluorescence in nuclei of Nicotiana benthamiana leaves 345 indicating nuclear localization of the heterodimer (Fig. 3d). No fluorescent signals could be detected 346 for the OsFD4–Hd3a interaction (Fig. 3e and Fig. S2). 347 348 OsFD4 can homodimerize or heterodimerize with OsFD3 349 bZIP transcription factors must homo- or heterodimerize to bind DNA (Dröge-Laser et al., 2018). 350 We thus tested combinatorial interactions between bZIPs. We previously showed that OsFD4 and 351 OsFD3 can form homo- and heterodimers, whereas no dimerization was found with OsFD1 352 (Brambilla et al., 2017; Fig. 3f,g). Here, we corroborated those results using BiFC, further showing 353 that OsFD4 and OsFD3 homo- and heterodimers are localized in the nucleus. We did not detect homo- 354 or heterodimerization of OsFD1 in BiFC assays (Fig. 3h,i). 355 356 OsFD4 regulates expression of AP1/FUL-like genes 357 The rice genome encodes four different AP1/FUL-like genes, OsMADS14, OsMADS15, OsMADS18 358 and OsMADS20 which control flower development (Wu et al., 2017). When plants are exposed to 359 inductive photoperiods and the meristem undergoes phase change, OsMADS14, OsMADS15 and 360 OsMADS18 increase their expression, whereas OsMADS20 is downregulated (Kobayashi et al., 2012; 361 Gómez-Ariza et al., 2019). In the SAM, OsFD1 promotes the expression of OsMADS14 and 362 OsMADS15. This regulation has been shown to be direct at least for OsMADS15, as the OsFD1- 363 containing FAC is able to bind to its promoter (Taoka et al., 2011). To assess if an OsFD4-containing 364 FAC could also regulate the expression of OsMADS genes we measured transcription of AP1/FUL- 365 like genes in apical meristems of osfd4-1. We performed time course experiments shifting plants from 366 non-inductive LD conditions to inductive SD conditions and sampled SAMs at 0, 6, 12 and 18 Days 367 After Shifting (DAS). These time points were chosen based on the progression of floral transition in 368 rice which becomes irreversible at 12 SDs, after which a fully committed SAM develops into a 369 branched inflorescence (Kobayashi et al., 2012; Gómez-Ariza et al., 2019). Comparing OsMADS 370 gene expression between wild type Dongjin and osfd4-1 plants, we observed that OsMADS14 and 371 OsMADS15 levels could increase in the mutant but at slower rates, whereas expression of OsMADS18 372 and OsMADS20 was not changed compared to the wild type (Fig. 4a-d). 11 373 Rice AP1/FUL-like genes work together with OsMADS34/PAP2, a SEPALLATA (SEP)-like gene, to 374 promote the floral transition (Kobayashi et al., 2010, 2012). Expression of OsMADS34/PAP2 375 increased in both wild type and osfd4-1 but was significantly delayed in the mutant (Fig. 4e). To 376 complete our analysis, we also quantified the expression of AP1/FUL-like genes and of 377 OsMADS34/PAP2 in the osfd1-1 mutant. We observed that their expression had similar dynamics in 378 both osfd1-1 and osfd4-1 (Fig. 4f-j). Taken together, these data suggest that OsFD4-FAC, as well as 379 OsFD1-FAC, act upstream of OsMADS genes to promote the rice floral transition. 380 381 Genome-wide identification of OsFD4, OsFD1 and HBF1 binding sites 382 In the SAM, changes in the photoperiod regulate the transcription of several genes (Furutani et al., 383 2006; Kobayashi et al., 2012; Tamaki et al., 2015; Gómez-Ariza et al., 2019). Yet, only a few genes 384 are known to be under control of FACs/FRCs. As a first screen to identify DNA binding sites of bZIP 385 transcription factors and FAC-regulated genes, we performed DNA Affinity Purification sequencing 386 (DAP-seq) using OsFD4, OsFD1 and HBF1 (O’Malley et al., 2016; Bartlett et al., 2017; Galli et al., 387 2018). Peak calling analysis identified 1107 DAP-peaks for OsFD4, 2059 DAP-peaks for OsFD1, 388 and 28323 DAP-peaks for HBF1 (Fig. 5a). The top enriched motif for OsFD1, OsFD4 and HBF1 389 contained the core consensus motif CACGT, the known binding site for many bZIP transcription 390 factors known as G-box (Fig. 5a) (O’Malley et al., 2016). At the flanks of this core motif, two 391 nucleotides -GC- were enriched with different frequencies, depending on the bZIP tested. For 392 example, OsFD4 peaks were most enriched for the motif GCCACGT (Fig. 5a). 393 To identify putative target genes regulated by these binding events, we assigned all DAP-peaks lying 394 within 5000 bp upstream of an ATG and 3000 bp downstream of a STOP codon (including regions 395 within exons and introns) to the closest gene. Based on this analysis, HBF1 peaks were associated 396 with 15,937 putative target genes, and OsFD1 and OsFD4 were associated with 1,717 and 925 397 putative target genes, respectively (Fig. 5a; Table S3). Most of the binding sites fell in putative 398 promoter regions (Fig. 5b). The highest frequency of peaks (30% for OsFD4, 27% for OsFD1 and 399 17% for HBF1) was in the 500 bp upstream of the transcription start site (TSS; Fig. 5c), consistent 400 with these sites being highly represented in the core promoter sequences. We next examined the 401 overlap between genes bound by OsFD1, OsFD4 and HBF1 and observed that 698 putative target 402 genes were shared by all tested bZIPs; 220 genes were shared by OsFD4 and HBF1, and 991 were 403 shared by OsFD1 and HBF1. All genes common to OsFD1 and OsFD4 were also common to HBF1, 404 thus no gene was found at the intersection between OsFD1 and OsFD4 datasets only (Fig. 5d and 405 Table S3). Finally, 7, 28 and 14,028 genes were uniquely targeted by OsFD4, OsFD1 or HBF1 (Fig. 406 5d). 12 407 The DAP-seq method uses the entire naked genome to identify transcription factor binding sites. 408 Thus, many bound regions might not be functionally relevant for regulation of gene expression in 409 vivo (O’Malley et al., 2016; Bartlett et al., 2017). To focus our analysis on those tissues where OsFD4, 410 OsFD1 and HBF1 are expressed (Fig. 2a), we filtered for genes expressed specifically in the SAM 411 and mature leaves. To this end, we first compared the lists of genes bound by OsFD1 and OsFD4 to 412 those expressed in the shoot apex, based on recently published RNA-seq data (Fig. 5e) (Gómez-Ariza 413 et al., 2019). The lists of genes bound by HBF1 and OsFD1 were compared to the list of genes 414 expressed in mature leaves, again as determined by published RNA-seq data (Galbiati et al., 2016) 415 (Fig. 5f). This analysis identified 240 genes expressed in shoot apices that were putative targets of 416 both OsFD4 and OsFD1, 58 genes that were putative targets of OsFD4, and 320 genes that were 417 putative targets of OsFD1 (Fig. 5e). Of those genes showing expression in mature leaves, 566 were 418 putative targets of both HBF1 and OsFD1, 3691 were putative targets of HBF1, and seven were 419 putative targets of OsFD1 (Fig. 5f). 420 Finally, we compared putative targets of OsFD1, OsFD4 and HBF1 with targets of AtFD (Collani et 421 al., 2019). To this end, genes identified by ChIP-seq data were retrieved from Collani et al. (2019) 422 and the corresponding putative rice orthologs were assigned based on the EnsemblPlants database 423 (Table S2). The overlap between corresponding genes indicates the existence of targets possibly being 424 part of common regulatory modules, evolutionary conserved (Fig. S4 and Table S4). 425 426 OsMADS62 and OsARF19 are targets of both the OsFD4-FAC and OsFD1-FAC 427 To determine if binding of OsFD1 and OsFD4 to genomic regions correlated with transcriptional 428 regulation of the neighboring genes, we quantified transcript abundance of selected target genes in 429 osfd1-1, osfd4-1 and wild type. For this analysis, we focused on several transcription factors 430 expressed in the apical meristem and whose promoters contained OsFD1 or OsFD4 binding peaks. 431 These included two genes bound by both OsFD1 and OsFD4, LOC_Os08g38590 (OsMADS62) and 432 LOC_Os06g48950 (OsARF19), three genes bound only by OsFD1, LOC_Os01g14440 (WRKY), 433 LOC_Os01g64360 (MYB), and LOC_Os04g51000 (RICE FLORICAULA/LEAFY), and two genes 434 bound by OsFD4 only, LOC_Os04g31730 (B3) and LOC_Os07g41580 (NF-YB) (Fig. 5g-h and Fig. 435 S5). Expression of both OsMADS62 and OsARF19 was reduced in osfd4-1 and osfd1-1 compared to 436 the wild type, suggesting that OsFD1 and OsFD4 may be direct activators of both genes (Fig. 5g-h). 437 The other genes tested showed reduced expression in both mutants relative to wild type probably 438 because of the epistatic effect of OsFD1 to OsFD4 (Fig. S5). These data suggest that OsFD4 and 439 OsFD1 regulate several common target genes, but their transcriptional effects are not completely 440 overlapping. 13 441 442 OsFD4, OsFD1 and HBF1 show preferential DNA binding configurations 443 OsFD1, OsFD4 and HBF1 bind overlapping but also distinct sets of genes in DAP-Seq experiments, 444 yet they recognize a very similar motif. Because bZIP TFs are known to bind to tandem motifs, we 445 asked if spacing between CACGT consensus motifs found in OsFD1, OsFD4 and HBF1 DAP-seq 446 peaks varied. We used a Position Weight Matrix (PWM) derived from DAP-seq data to locate bZIP 447 binding sites and test whether there are some preferences in terms of binding syntaxes (spacing and 448 configuration between two consensus motifs (Fig. 6a) (Stigliani et al., 2019). We first evaluated the 449 predictive power of the generated PWM which resulted exceptionally high, with ROC values of 450 0.985, 0.971 and 0.9 respectively (Fig. S6). Next, we analyzed the distribution of spacings for each 451 bZIP, quantifying overrepresented configurations against a set of negative regions (unbound), and we 452 found specific profiles for each bZIP. 453 Direct Repeats (DR) 0, Everted Repeats (ER) 1, 5, ER18-20, ER44, Inverted Repeats (IR) 41 454 configurations showed at least a 4-fold overrepresentation among OsFD4 bound regions, with the 455 ER44 configuration being highly overrepresented (Fig. 6b). Regions bound by OsFD1 showed a 456 robust enrichment of at least 3-fold in the DR30, ER4, ER16, ER32-34, ER41-2 and ER46, and IR38 457 configurations (Fig. 6c), with ER16 and ER32-34 as the most overrepresented. Configuration 458 enrichments for HBF1 included DR29, DR40, ER3, ER16-18, ER42 and IR10 with at least 2-fold 459 enrichment compared to the negative set of regions (Fig. 6d). These data identify different spacing 460 configurations between consensus motifs, which are preferred by OsFD4, OsFD1 and HBF1 and 461 likely contribute to the specificity of target recognition. 462 463 DISCUSSION 464 Transcription factors of the bZIP family have important roles during phase transitions in different 465 plant species (Abe et al., 2005; Wigge et al., 2005; Taoka et al., 2011; Tsuji et al., 2013; Park et al., 466 2014; Tylewicz et al., 2015; Brambilla et al., 2017; Teo et al., 2017). However, those controlling 467 flowering time and for which functional studies have been carried out, appear insufficient to account 468 for all aspects of phase change, suggesting a certain level of redundancy, or the existence of additional 469 transcriptional regulators. In rice, the role of OsFD1 as a promoter of flowering has been supported 470 by overexpression studies, but the analysis of RNAi mutants failed to demonstrate phenotypic effects 471 on flowering (Taoka et al., 2011; Jang et al., 2017; Brambilla et al., 2017). Other OsFD-like genes 472 have been investigated previously, however they perform functions distinct from those of OsFD1 473 (Tsuji et al., 2013; Brambilla et al., 2017). Here, to identify additional components of florigen 474 signalling, we reported the characterization of OsFD4 and OsFD3, as well as further insights into 14 475 OsFD1 function based on CRISPR-mediated mutagenesis. While describing such additional FD-like 476 components integrating florigen signalling, it became clear that despite similar modes of action, 477 differences in their expression profiles, specific combinatorial interactions with Gf14 proteins and 478 florigens, and distinct DNA binding syntaxes distinguish different members of the family. 479 480 The FAC paradigm across Angiosperms 481 Formation of a FAC requires that florigenic proteins that have reached the SAM first interact in the 482 cytosol with Gf14 proteins and then enter the nucleus, where phosphorylated bZIP transcription 483 factors contact the Gf14 and contribute a DNA-recognition function. This model of interaction 484 between florigens and bZIP transcription factors was first proposed in Arabidopsis and later supported 485 by studies in rice, where the role of Gf14s as bridges was identified. Subsequently, it has been 486 extended to many other species including dicots and monocots and shown to control a wide range of 487 developmental processes. 488 In rice, the functional homolog of Arabidopsis FD is encoded by OsFD1. When overexpressed using 489 either a constitutive or phloem-specific promoter, OsFD1 can accelerate flowering (Jang et al., 2017, 490 Brambilla et al., 2017). However, RNAi lines flower similarly to wild type, possibly OsFD1 491 transcripts are not completely absent in these knock-down lines (Taoka et al., 2011). Thus, the 492 functional significance of OsFD1 as a flowering regulator has remained unclear. Knock-out of OsFD1 493 using CRISPR-Cas9 showed that the gene has indeed a prominent effect in promoting flowering. This 494 is likely the result of a more complex molecular function that includes (i) induction of OsMADS14 495 and 15 gene expression in the SAM and in leaves, (ii) induction of Ehd1, Hd3a and RFT1 mRNA 496 expression in leaves, and (iii) interaction with RCNs in repressor complexes that antagonize FAC- 497 dependent activation of OsMADS gene expression. Thus, OsFD1 and Gf14 proteins are central to 498 both activating and repressive flowering pathways, the switch between them being likely controlled 499 by the relative abundance of FT-like and TFL1-like proteins at the apex. 500 Still, while double Hd3a and RFT1 RNAi mutants do not flower for up to 300 days under inductive 501 SDs, single osfd1 loss-of-function mutants are delayed in flowering but they can flower, an apparent 502 inconsistency justified by functional redundancy between bZIPs (Komiya et al., 2008). In 503 Arabidopsis, FD is redundant with FD PARALOG (FDP). Double fd fdp mutations flower later than 504 fd single, and almost completely suppress precocious flowering of 35S:FT (Jaeger et al., 2013). In 505 rice, by mutating additional bZIPs, including OsFD3 and OsFD4, we have identified floral promoting 506 factors. Mutations in osfd4, but not in osfd3, can delay flowering and activation of OsMADS targets. 507 However, flowering was not delayed as much as in osfd1 mutants, indicating a less prominent role 508 compared to OsFD1. Also, osfd1 osfd4 double mutants did not further delay flowering compared to 15 509 osfd1 single mutants. Based on expression data, we propose that OsFD1 activates OsFD4 510 transcription. Thus, in osfd1 mutants, the levels of both regulators are low. This arrangement does not 511 fully exclude redundancy but suggests a complex regulatory network. 512 Considering the interaction patterns with the florigens, we found that OsFD4 can interact directly 513 with RFT1, but only via Gf14s with Hd3a, suggesting different preferences for FAC architectures. 514 The SAM of rice is induced to flower by both florigens under SD, but only by RFT1 under LD 515 (Komiya et al., 2008, 2009). Since the flowering delay observed in osfd4 mutants is much stronger 516 under LD than under SD, we propose that different bZIPs perform distinct functions in the 517 photoperiod, and that the OsFD4-RFT1 module is key to promote flowering mostly under non- 518 inductive conditions. 519 520 DAP-Seq identifies genome-wide OsFD1, OsFD4 and HBF1 binding sites 521 Application of DAP-seq using purified OsFD1, OsFD4 and HBF1 proteins resulted in the 522 identification of hundreds of putative direct binding sites for these bZIPs. Consistent with a function 523 in core promoters, binding sites were enriched in the proximity of the TSS. It should be noted that the 524 in vitro nature of DAP-seq allows for the identification of direct binding events by a single TF, 525 specifically capturing those mediated by individual homodimers. Given that OsFD1, OsFD4 and 526 HBF1 formed heterodimers, these events may represent only a fraction of all in vivo binding events 527 resulting from additional protein interactions. Integrating DAP-seq data with expression data allowed 528 us to focus specifically on those genes that were expressed in relevant tissues and on those 529 differentially expressed in the osfd4-1 and osfd1-2 mutants. Our data provide a first examination of 530 the direct genome-wide binding landscape of bZIP flowering regulators in rice. This approach needs 531 now to be refined for several reasons. 532 First, we used a single purified bZIP transcription factor in DAP-seq reactions. Being disengaged 533 from its florigen-containing complex, its DNA binding capacity might be biased. Arabidopsis FD can 534 bind functional sites without assembling into a FAC and with no need for FT or its closest homologue, 535 TWIN SISTER OF FT (TSF) (Collani et al., 2019). Also purified OsFD1 alone can bind a fragment 536 of the AP1 promoter containing a C-box element (GACGTC) in gel shift assays (Taoka et al., 2011). 537 We previously showed that HBF1 can bind in vitro an ABRE/G-box element in the Ehd1 promoter 538 region, a finding that we further corroborated in this study (Brambilla et al., 2017). Thus, bZIPs are 539 likely to conserve their basal DNA binding ability even when not combined in ternary complexes, 540 but despite these examples, affinity for DNA is likely potentiated by binding with other interactors, 541 including members of the PEBP family (Collani et al., 2019). 16 542 Second, since bZIPs bind the DNA as homo or heterodimers, and given their combinatorial 543 interactions and overlapping expression in different plant tissues, including the SAM, it cannot be 544 excluded that DNA binding specificity changes when assessing heterodimeric configurations. 545 Third, bZIPs might be incorporated into higher order complexes that include other transcriptional 546 regulators with different DNA binding properties. In Arabidopsis, SQUAMOSA PROMOTER- 547 BINDING PROTEIN-LIKE 3 (SPL3), SPL4 and SPL5 interact with FD to enhance its transcriptional 548 activation of target genes and its specificity for target promoters (Jung et al., 2016). Since SPLs are 549 produced at the SAM in response to plant aging, the SPL-FD complex has been proposed to integrate 550 photoperiodic and aging signals to activate the AP1 promoter (Jung et al., 2016). The study further 551 suggested that the proximal C-box in the AP1 promoter is dispensable for FD-mediated induction, 552 and that a distinct SPL-binding site might contribute to transcription of AP1. A similar case has been 553 described for Class II TCP transcription factors, including TCP5, 13 and 17. These proteins can 554 physically interact with FD, and facilitate its binding to the AP1 promoter. Also in this case, binding 555 of FD to the AP1 promoter was not observed at the C-box, but was dependent upon a TCP binding 556 motif located between the SPL-binding site and the C-box (Li et al., 2019). 557 Finally, post transcriptional modifications have a major role in vivo. Many FD-like proteins harbour 558 at their C terminus an SAP domain containing serine, threonine or both, which needs to be 559 phosphorylated to interact with Gf14s (Taoka et al., 2011; Park et al., 2014; Li et al., 2015; Collani 560 et al., 2019). When phosphorylated, Arabidopsis FD has a higher affinity for DNA. Expression of 561 phosphomimic versions of FD or OsFD1 can accelerate flowering in Arabidopsis and rice, 562 respectively. The kinases responsible for phosphorylation in Arabidopsis include Calcium-dependent 563 Protein Kinase 6 (CPK6) and CPK33, which interact with FD and, if mutated, slightly delay flowering 564 (Kawamoto et al., 2015). Thus, optimal DNA binding and floral promoting activity of FAC 565 complexes likely relies on this modification of the bZIP components. Future DNA-binding 566 experiments either in vivo or using multiple complex components could help elucidate the individual 567 contributions of each component. 568 569 Direct targets of OsFD1 and OsFD4 and alternative binding syntaxes 570 Despite these caveats, DAP-seq led to the identification of several putative direct targets of the bZIPs 571 studied here, out of which OsMADS62 and ARF19 were validated as transcriptionally regulated by 572 both OsFD1 and OsFD4. OsMADS62 controls pollen maturation and germination, partially 573 redundantly with OsMADS63 and OsMADS68 (Liu et al., 2013). OsFD1 and OsFD4 promoted 574 OsMADS62 expression, a function they likely perform after meristem commitment, during 575 inflorescence and flower development. OsARF19 encodes for an AUXIN RESPONSE FACTOR 17 576 broadly expressed in the plant but with higher levels of transcription in the shoot, which is further 577 elevated by auxin treatments (Zhang et al., 2015). 578 We did not find OsFD1 or OsFD4 bound to the OsMADS14, OsMADS15 or OsMADS34/PAP2 579 promoter. OsMADS15 has been proposed as direct target of an OsFD1-containing FAC (Taoka et al., 580 2011). Other proteins could possibly stabilize OsFD4 and OsFD1 in vivo and allow binding to these 581 loci. Alternatively, the rice OsFD1-OsMADS connection might be indirect. Data in support of a direct 582 connection have not been as thoroughly repeated and validated in rice as they have been in 583 Arabidopsis, in which direct contacts between FD and the C-box element in the AP1 promoter were 584 disproved (Benlloch et al., 2011; Jung et al., 2016; Collani et al., 2019; Li et al., 2019). Further 585 assessment of in vivo binding will be necessary. 586 Genome scanning by DAP-seq identified identical core bZIP-binding motifs that were highly 587 enriched in peaks of all three bZIP datasets. The same 5’-CACGT-3’ core motif was identified as 588 bound by OsFD1, OsFD4 and HBF1. The same 5’-CACGT-3’ core motif was identified as enriched 589 among FD binding regions in vivo, suggesting conservation between rice and Arabidopsis, and indeed 590 many other bZIPs from diverse organisms (Dröge-Laser et al., 2018). 591 We found that despite identical core DNA binding sites, spacing among tandem motifs was different 592 for each of the three bZIPs tested in our assay. OsFD1 and OsFD4 which are activators of the rice 593 floral transition prefer to bind the DNA with ER conformations, whereas HBF1, a repressor, binds 594 more frequently ER and DR conformations. These results indicate distinct and specific binding 595 syntaxes for each transcription factor. The method was recently applied to define binding syntaxes of 596 MONOPTEROS (MP) and AtARF2, which have opposing transcriptional functions but share the 597 same consensus motif (Stigliani et al., 2019). The method can capture DNA binding features of 598 proteins that interact as either homo or heterodimers, but the ER or DR arrangements might suggest 599 the possibility of more elaborated structures, including tetramers. 18 600 FIGURE LEGENDS 601 602 Figure 1. The osfd4 and osfd1 mutants delay rice flowering. 603 (a) Days to heading of Dongjin wild type (Dj) and osfd4-1 under inductive short day conditions (SD) 604 and non-inductive long day conditions (LD). (b) Days to heading of Nipponbare wild type (Nb), 605 osfd1-1, osfd3-1 and osfd4-3 under SD conditions. (c) Representative pictures of osfd1-1 (left) and 606 Nb (right) plants. White arrows indicate emerging panicles. (d) Days to heading of Nb, osfd1-2, Dj, 607 osfd4-1 and double osfd4-1 osfd1-2 mutants under SD conditions. Data are represented as mean ± 608 StDev. Asterisks indicate the p-value calculated using ANOVA, ****=p<0.0001, **=p<0.01, ns=non 609 significant. All flowering time experiments were repeated at least twice, and one representative 610 experiment is shown in (a) and (b). The graph in (d) includes data from three independent 611 experiments. 612 613 Figure 2. Temporal expression pattern of OsFD1, OsFD3 and OsFD4 in the shoot apical 614 meristem of rice and genetic interaction between OsFD1 and OsFD4. 615 Quantification of OsFD1, OsFD3 and OsFD4 transcripts in the SAM (a) and in leaves (b) of Nb wild 616 type. DAS, Days After Shift from long day (LD) to short day (SD) conditions. (c) Quantification of 617 OsFD4 transcripts in SAMs of osfd1-1 mutants. (d) Quantification of OsFD1 transcripts in SAMs of 618 osfd4-1 mutants. Each time point represents the mean ± StDev of three technical replicates. The 619 experiments were repeated twice with similar results. Ubiquitin was used to normalize gene 620 expression. 621 622 Figure 3. Interactions between OsFD1, OsFD3, OsFD4 and components of Florigen Activation 623 Complexes (FACs) determined in yeast and tobacco. 624 (a) Interactions between Activation Domain (AD)-OsFD1, AD-OsFD4 and Binding Domain (BD)- 625 Gf14A-F. (b) Interactions between AD-Gf14A-F and BD-Hd3a and BD-RFT1. (c) Interactions 626 between AD-OsFD4, AD-OsFD3 and Hd3a and RFT1 fused to the BD. Bimolecular fluorescence 627 complementation (BiFC) between OsFD4 fused with N-terminus of YFP (N-YFP) and (d) RFT1 628 fused with the C-terminus of YFP (C-YFP) or (e) Hd3a fused with the C-YFP. (f) Interactions 629 between OsFD4 and OsFD1 fused with the AD or the BD. (g) Interactions between OsFD3, OsFD4 630 and OsFD1 fused to the BD and OsFD3 fused to the AD. (h) Assessment of OsFDs homodimerization 631 by BiFC. From left, OsFD4:N-YFP/OsFD4:C-YFP, OsFD1:N-YFP/OsFD1:C-YFP and OsFD3:N- 632 YFP/OsFD3:C-YFP. (i) Assessment of OsFDs heterodimerization by BiFC. From left OsFD4:N- 633 YFP/OsFD1:C-YFP, OsFD3:N-YFP/OsFD4:C-YFP and OsFD1:N-YFP/OsFD3:C-YFP. 19 634 Interactions were determined in yeast on selective drop out media -L-W-H-A. The yeast experiments 635 were repeated three times with identical results. AD and BD clones containing empty vectors were 636 used as negative controls. BiFC experiments were repeated three times in tobacco with identical 637 results. N-YFP and C-YFP clones containing empty vectors were used as negative controls. DAPI 638 stain was used to mark nuclei. 639 640 Figure 4. OsFD4 and OsFD1 promote OsMADS14 and 15 transcription in rice. 641 Quantification of (a), (f) OsMADS14, (b), (g) OsMADS15, (c), (h) OsMADS18, (d), (i) OsMADS20, 642 and (e), (j) OsMADS34/PAP2 transcription in Dongjin vs. osfd4-1 mutants (a)-(e) and in Nipponbare 643 vs. osfd1-1 mutants (f)-(j). Expression was quantified in plants grown for two months under long day 644 (LD) and then shifted to short day (SD) conditions. Apical meristems were sampled at 0, 6, 12 and 645 18 Days After Shifting (DAS). 646 647 Figure 5. Identification of OsFD1, OsFD4 and HBF1 binding sites. 648 (a) Summary of DNA Affinity Purification sequencing (DAP-seq) results of OsFD1, OsFD4 and 649 HBF1, including number of peaks, putative target genes and consensus motives. (b) Distribution of 650 peaks within gene features. (c) Distribution of peaks near the Transcriptional Start Site (TSS) of 651 putative target genes. (d) Venn diagrams showing the overlap between putative target genes of 652 OsFD1, OsFD4 and HBF1. (e) Venn diagrams showing the overlap between targets of OsFD1 and 653 OsFD4 and genes expressed at the apical meristem. (f) Venn diagrams showing the overlap between 654 targets of OsFD1 and HBF1 and genes expressed in leaves. Genome browser view of OsFD1 and 655 OsFD4 binding peaks in the 3’UTR of OsMADS62 (g) and in the putative promoter of OsARF19 (h), 656 and quantification of transcripts in osfd4-1 and osfd1-1 mutants. Black arrows indicate the direction 657 of transcription. Asterisks indicate p<0.01 (*), p<0.005 (**), p<0.001 (***), p<0.0001 (****) based 658 on Student’s t test, ns=non significant. Each time point represents the mean ± St Dev of three technical 659 replicates. The experiments were repeated twice with similar results. Ubiquitin was used to normalize 660 gene expression. 661 662 Figure 6. Analysis of OsFD1, OsFD4 and HBF1 binding syntax. 663 (a) Scheme of Everted Repeats (ER), Direct Repeats (DR) and Inverted Repeats (IR) of “ACGTGGC” 664 motives. Blue and green arrows represent the first and the second binding motifs. N=general base; 665 n=number of base pairs between the two motifs. (b)-(d) Spacing analysis of absolute and normalized 666 enrichment of OsFD4 (b), OsFD1 (c) and HBF1 (d) binding sites. The numbers on top of the graphs 667 represent the distance between two consensus motives. 20 668 669 Supplemental figures and tables 670 Fig. S1 Genotype and phenotype of OsFD4 and OsFD1 mutants 671 Fig. S2 OsFD3 and Gf14s Y2H assays and OsFD4-Hd3a BiFC 672 Fig. S3 Y2H mating controls 673 Fig. S4 Venn diagram showing the overlap between targets of AtFD, OsFD1, OsFD4 and HBF1 674 Fig. S5 Expression analysis of some DAP-seq targets in osfd4-1 and osfd1-1 675 Fig. S6 Spacing analysis control: OsFD4, OsFD1 and HBF1 ROC curves 676 Table S1 Sequences of oligonucleotides used in this study 677 Table S2 Arabidopsis orthologs of rice genes 678 Table S3 List of OsFD4, OsFD1 and HBF1 DAP-seq bound genes 679 Table S4 List of genes shown in Figure S4 680 681 Acknowledgements 682 The authors wish to thank the NOLIMITS microscopy facility and Botanical Garden Città Studi for 683 technical support. This work was supported by an ERC Starting Grant (#260963) and PRIN 684 (#20153NM8RM) to FF. 685 686 Author Contributions 687 MC, MG, JL, F Parcy, AG, VB and FF designed research; MC, FG, MG, BK, JL and F Podico 688 performed research; MC, FG, MG, BK, JL, ET, VB and FF analysed and interpreted data; MC and 689 FF wrote the manuscript. 690 691 REFERENCES 692 Abe M, Kobayashi Y, Yamamoto S, Daimon Y, Yamaguchi A, Ikeda Y, Ichinoki H, Notaguchi 693 M, Goto K, Araki T. 2005. FD, a bZIP Protein Mediating Signals from the Floral Pathway 694 Integrator FT at the Shoot Apex. Science 309: 1052–1056. 695 Andrés F, Coupland G. 2012. The genetic basis of flowering responses to seasonal cues. Nature 696 Reviews. Genetics 13: 627–39. 697 Bartlett A, Malley RCO, Huang SC, Galli M, Nery JR, Gallavotti A, Ecker JR. 2017. Mapping 698 genome-wide transcription-factor binding sites using DAP-seq. Nature Protocols 12: 1659–1672. 699 Benlloch R, Kim MC, Sayou C, Thévenon E, Parcy F, Nilsson O. 2011. 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Journal of Experimental Botany 68: 553–568. 27 (a) (b) ✱✱✱✱ ✱✱✱✱ 50 200 160 Days to Haeding Days to Haeding 180 140 ✱✱✱✱ 60 50 40 30 ✱✱✱✱ ✱✱✱✱ 20 40 D D jL 1 4fd os LD N (d) b 1 1 3 134fd fd fd os os os ns 70 60 50 ✱✱ 40 ✱✱✱✱ ✱✱✱✱ 30 D os j fd 1/ 4os 1 fd D 1j 2 N bx D j SD os fd 4- 1 4fd os N os b fd 12 N b (c) D jS Days to Haeding D (a) (b) SAM OsbZIPs/UBQ 4×10 -1 LEAVES 2×10 -1 1×10 -1 2×10 -1 4×10 -3 2×10 -3 5×10 -4 1×10 -3 0 0.006 0 0 6 12 18 Days after Shift (DAS) Nb wt 0 6 12 18 Days after Shift (DAS) (d) 0.4 osfd1-1 OsFD1/UBQ (c) OsFD4/UBQ OsFD4 OsFD1 OsFD3 3×10 -3 1×10 -3 0.004 0.002 Dj wt osfd4-1 0.3 0.2 0.1 0.0 0.000 0 12 Days after Shift (DAS) 0 12 Days after Shift (DAS) BD empty vector AD::Gf14A BD::Gf14A AD::Gf14B BD::Gf14B AD::Gf14C BD::Gf14C AD::Gf14D BD::Gf14D AD::Gf14E BD::Gf14E AD::Gf14F BD::Gf14F AD empty vector BD empty vector -W, -L, -H, -A BD empty vector BD::RFT1 BD::Hd3a -W, -L, -H, -A (c) BD::RFT1 BD::Hd3a (b) AD empty vector AD::OsFD4 AD::OsFD1 (a) (d) OsFD4:N-YFP RFT1:C-YFP (e) OsFD4:N-YFP Hd3a:C-YFP AD::OsFD4 AD::OsFD3 AD empty vector AD empty vector (g) AD::OsFD3 AD empty vector AD::OsFD1 (f) AD::OsFD4 -W, -L, -H, -A BD::OsFD3 BD::OsFD4 BD::OsFD4 BD::OsFD1 BD::OsFD1 BD empty vector BD empty vector -W, -L, -H,-A -W, -L, -H, -A (h) OsFD4:N-YFP OsFD4:C-YFP OsFD1:N-YFP OsFD1:C-YFP OsFD3: N-YFP OsFD3:C-YFP (i) OsFD4:N-YFP OsFD1:C-YFP OsFD3:N-YFP OsFD4:C-YFP OsFD1:N-YFP OsFD3:C-YFP (a) (f) OsMADS14/UBQ 1.2×10 0 1.2×10 0 Dj osfd1-1 8×10 -1 8×10 -1 4×10 -1 4×10 -1 0 0 0 6 12 18 OsMADS15/UBQ (b) 1.26×10 0 1.26×10 0 8.4×10 -1 8.4×10 -1 4.2×10 -1 4.2×10 -1 6 12 18 OsMADS18/UBQ (c) 3×10 -1 3×10 -1 2×10 -1 2×10 -1 1×10 -1 1×10 -1 18 0 6 12 18 0 6 12 18 0 6 12 18 0 6 12 18 0 0 OsMADS20/UBQ 12 (h) 0 6 12 18 (i) 1.8×10 -2 1.8×10 -2 1.2×10 -2 1.2×10 -2 6×10 -3 6×10 -3 0 0 0 OsMADS34/UBQ 6 0 0 (e) 0 (g) 0 (d) Nb osfd4-1 6 12 18 (j) 5.7×10 -2 5.7×10 -2 3.8×10 -2 3.8×10 -2 1.9×10 -2 1.9×10 -2 0 0 0 6 12 18 Days after Shift (DAS) Days after Shift (DAS) OsFD1 OsFD4 (b) OsHBF1 (a) bZIP Nr of Nr of putative peaks target genes HBF1 28323 15937 OsFD1 2059 1717 OsFD4 1107 925 Upstream 5' UTR Exons/CDS Introns 3' UTR Downstream Unclassified Motif Peak Frequency (c) 6 OsFD1 OsFD4 OsHBF1 4 2 0 Distance from TSS (bp) (d) (e) (f) OsFD4 HBF1 HBF1 Leaves genes OsFD4 OsFD1 OsFD1 SAM genes OsFD1 (h) (g) OsMADS62 Dj osfd4-1 OsMADS62/UBQ * ** Nb osfd1-1 2×10 -3 ns * * 1.5×10 -3 1.5×10 -3 1×10 -3 1×10 -3 5×10 -4 5×10 -4 ** Dj osfd4-1 *** * 0 0 0 6 12 Days after Shift (DAS) 18 8×10 -2 OsARF19/UBQ 2×10 -3 OsARF19 0 6 12 Days after Shift (DAS) 18 ns *** Nb osfd1-1 ** ** 2×10 -2 6×10 -2 1.5×10 -2 4×10 -2 1×10 -2 2×10 -2 5×10 -3 0 ns * ns **** 0 6 12 18 0 0 6 12 Days after Shift (DAS) 18 Days after Shift (DAS) (a) (b) 44 34 1-5 0 41 18-20 (c) 16 32-34 38 4 30 28 41-46 (d) 29 40 10 18 42 16-18 3 29 10 New Phytologist Supporting Information Article title: OsFD4 promotes the rice floral transition via Florigen Activation Complex formation in the shoot apical meristem Authors: Martina Cerise, Francesca Giaume, Mary Galli, Bahman Khahani, Jérémy Lucas, Federico Podico, Elahe Tavakol, François Parcy, Andrea Gallavotti, Vittoria Brambilla and Fabio Fornara Article acceptance date: 15 July 2020 The following Supporting Information is available for this article: Fig. S1 Genotype and phenotype of OsFD4 and OsFD1 mutants Fig. S2 OsFD3 and Gf14s Y2H assays and OsFD4-Hd3a BiFC Fig. S3 Y2H mating controls Fig. S4 Venn diagram showing the overlap between targets of AtFD, OsFD1, OsFD4 and HBF1 Fig. S5 Expression analysis of some DAP-seq targets in osfd4-1 and osfd1-1 Fig. S6 Spacing analysis control: OsFD4, OsFD1 and HBF1 ROC curves Table S1 Sequences of oligonucleotides used in this study Table S2 Arabidopsis orthologs of rice genes (see separate file) Table S3 List of OsFD4, OsFD1 and HBF1 DAP-seq bound genes (see separate file) Table S4 List of genes shown in Figure S4 (see separate file) (a) osfd4-1 (b) (d) OsFD4/UBQ 0,004 BASIC REGION LEUCINE ZIPPER 0,003 0,002 SAP DOMAIN LEUCINE-ZIPPER 0,001 0 Dj Wt osfd4-1 (e) OsFD4 osfd4-3 osfd4-4 osfd4-5 OsFD1 osfd1-1 osfd1-2 OsFD3 osfd3-1 osfd3-2 55 | CCGCCGGCGTCGTTTC-------------------AGGACGG CCGCCGGCGTCGT---CATCGTCCCGCCGGCGACGAGGACGG CCGCCGGCGTCG----C-------------------GGACGG CCGCCGGCGTCGTTTC------------------AAGGACGG 367 | GCCGGGGACCGCCGCAA-GAGGCGGATGATCAAGAACCGCGA GCCGGGGACCGCCGCAATGAGGCGGATGATCAAGAACCGCGA GCCGGGGACCGCCGCAA--AGGCGGATGATCAAGAACCGCGA 87 | CAGCGCCGCCGATGGTGCTCCCCATCTATCCCTCGGCGTGGG CAGCGCCGCCGATGGTGCTCCCCATCTA--CCTCGGCGTGGG CAGCGCCGCCGATGGTGCTCCCCATC-ATCCCTCGGCGTGGG ✱✱✱ 180 WT + 16 -4 +1 WT +1 -1 WT -2 -1 Heading Days (c) ✱✱ 160 140 120 100 o d sf 4 4o d sf 3 4- N b t w Fig. S1 (a) Map of the osfd4-1 mutant locus. Green and blue lines represent exons and UTR regions, respectively. A black line indicates an intron. A triangle in the 3’UTR region indicates the position of the T-DNA insert. (b) Expression of OsFD4 in the SAM of Dj wild type and osfd4-1 mutant. Data are average ± StDev of three technical replicates. (c) Sequences of mutant alleles of osfd4, osfd1 and osfd3 obtained using CRISPR. Inserted nucleotides are indicated in green. (d) Protein alignment of the C-terminus region of OsFD1, OsFD4 and OsFD3 starting from the basic leucine zipper domain. (e) Flowering time under LD conditions of osfd4-3 and osfd4-4. Data are represented as mean ± StDev. Asterisks indicate the p-value calculated using ANOVA, AD empty vector (a) AD::OsFD3 ***=p<0.001, **=p<0.01. (b) BD::Gf14A BD::Gf14B BD::Gf14C BD::Gf14D BD::Gf14E (c) BD::Gf14F (d) BD empty vector -W, -L, -H, -A OsFD4: C-YFP Gf14B N-YFP Empty vector C-YFP OsFD4: N-YFP Hd3a: C-YFP Empty vector N-YFP Fig. S2 (a) Yeast two Hybrid (Y2H) among Gf14A-F fused to the Binding Domain (BD) and OsFD3 fused to the Activation Domain (AD). (b) Double entry table summarizing homo- and heterodimerizations among Gf14A-F. Interactions were assessed on drop out media lacking L, W, H and A. N.T., not tested. (c) Bimolecular fluorescence complementation (BiFC) between OsFD4 fused with C-terminus of YFP (C-YFP) and Gf14B fused with the N-terminus of YFP (NYFP). (d) BiFC between empty C-YFP vector and OsFD4 fused with N-YFP (left); Hd3a fused with C-YFP and N-YFP empty vector (right). Negative controls were done for every independent BiFC assay performed; here representative ones are shown. Fig. S3 (a) Mating controls of the Y2H among Gf14A-F fused to the AD and Hd3a or RFT1 fused to the BD. (b) Mating controls of the Y2H among Gf14A-F fused to the BD and OsFD1, OsFD4 and OsFD3 fused to the AD. (c) Mating controls of the Y2H among OsFD4 and OsFD3 fused to the AD and Hd3a and RFT1 fused to the BD. (d) Mating controls of the Y2H among OsFD4 and OsFD1 fused with the AD or the BD. (e) Mating controls of the Y2H among OsFD3, OsFD4 and OsFD1 fused to the BD and OsFD3 fused to the AD. All controls were grown on yeast medium lacking W and L. Fig. S4 Venn diagram showing the overlap between targets of AtFD, OsFD1, OsFD4 and HBF1. (a) Dj osfd1-1 3×10 -3 LOC_Os01g14440/UBQ LOC_Os01g14440/UBQ Nb osfd4-1 3×10 -3 2×10 -3 1×10 -3 2×10 -3 1×10 -3 0 0 (b) 12 0 18 0 8×10 -3 LOC_Os01g64360/UBQ LOC_Os01g64360/UBQ 6 Days after Shift (DAS) 5×10 -3 4×10 -3 3×10 -3 2×10 -3 1×10 -3 18 4×10 -3 2×10 -3 0 0 6 12 0 18 Days after Shift (DAS) 6 12 18 Days after Shift (DAS) 6×10 -2 1.5×10 -2 LOC_Os04g51000/UBQ LOC_Os04g51000/UBQ 12 6×10 -3 0 (c) 6 Days after Shift (DAS) 4×10 -2 2×10 -2 1×10 -2 5×10 -3 0 0 0 6 12 0 18 Days after Shift (DAS) 6 12 18 Days after Shift (DAS) (d) 4×10 -3 LOC_Os04g31730/UBQ LOC_Os04g31730/UBQ 2.5×10 -2 2×10 -2 1.5×10 -2 1×10 -2 5×10 -3 0 3×10 -3 2×10 -3 1×10 -3 0 0 (e) 6 12 0 18 Days after Shift (DAS) 1×10 -2 12 18 2×10 -3 LOC_Os07g41580/UBQ LOC_Os07g41580/UBQ 6 Days after Shift (DAS) 8×10 -3 1.5×10 -3 6×10 -3 4×10 -3 2×10 -3 0 1×10 -3 5×10 -4 0 0 6 12 Days after Shift (DAS) 18 0 6 12 18 Days after Shift (DAS) Fig. S5 Quantification of transcripts of LOC_Os01g14440 (WRKY) (a), LOC_Os01g64360 (MYB) (b), LOC_Os04g51000 (RICE FLORICAULA/LEAFY) (c), LOC_Os04g31730 (B3) (d) and LOC_Os07g41580 (NF-YB) (e) in osfd4-1 and osfd1-1 mutants. Data are represented as mean ± StDev. Fig. S6 Receiver Operating Characteristics (ROC) analysis of OsFD4 (a), OsFD1 (b) and HBF1 (c) estimating the prediction power of Position Weight Matrices (PWMs), i.e. the capability of OsbZIPs binding sites to differentiate between bound and not bound regions. AUC, Area Under the Curve. Table S1 Sequences of oligonucleotides used in this study. Number Sequence Gene Os_892 Os_893 Os_245 Os_246 Os_160 Os_161 Os_158 Os_159 Os_239 Os_240 Os_353 Os_354 Os_1353 Os_1354 Os_186 Os_187 Os_1312 Os_1313 Os_1341 Os_1342 Os_1349 Os_1350 Os_1351 Os_1352 Os_1369 Os_1370 Os_1371 Os_1372 Os_1373 Os_1374 Os_1151 Os_1152 Os_936 Os_937 Os_1153 Os_1154 GACAACGTGAAGGCGAAGA CACCAGGTGGAGTGTGGAC CCGGGCTATGGAGTGAAAT TGCTCTCATATTCTCCATGCTG TTCAGGTGGACGACCTTAGC GCTAGCAGCTGCCAACACT CGGTTGCGAGACGAGGAA GAAAGACGGTGCTGGACGAA CGTCGTCGGCCAAACAG TGACTTCAATTCATTCAAGGTTGCT TGTTCTGCAAAAGCTCATGG TTGGTGATGATGTGCTTGGT GCTCCAGTCATCATCGAACG GGAGATACTACTACCGCTCACT TTGATGAACTCTGCGACCTAAA TGCTGCAGTTTCCGTTCC AGAGGGGCAACTTGAGGAAC GCAACCGACAAATTCCTCC CAGTACCTGTCGATGGAGCA GTTGCTGTCGTACGCCATG GCAACACCAACAACACAAGC AGTGTATACCATGTGCCCACT GCATCCATGACATCACCACC ATCAGAGAGCCCCAAGTGAC GTTCAGGGTGCAGGTCCTC ACCTGAAACTGAAACCTCCCA ATGGTGATGGGAGGGAAAGG ACCCGACATGTCCTCTGATT GTACGTGCCCACCAGACT GCCCAACACCACAGGAAAC GGCACCGCCGGCGTCGTTTCAGGA AAACTCCTGAAACGACGCCGGCGG GGCATCTTGATCATCCGCCTCTTG AAACCAAGAGGCGGATGATCAAGA GGCAGCCCACGCCGAGGGATAGAT AAACATCTATCCCTCGGCGTGGGC UBQ Fw for qRT-PCR UBQ Rv for qRT-PCR OsFD4 Fw for qRT-PCR OsFD4 Rv for qRT-PCR OsFD1 Fw for qRT-PCR OsFD1 Rv for qRT-PCR OsMADS14 Fw for qRT-PCR OsMADS14 Rv for qRT-PCR OsMADS15 Fw for qRT-PCR OsMADS15 Rv for qRT-PCR OsMADS18 Fw for qRT-PCR OsMADS18 Rv for qRT-PCR OsMADS20 Fw for qRT-PCR OsMADS20 Rv for qRT-PCR OsMADS34 Fw for qRT-PCR OsMADS34 Rv for qRT-PCR OsARF19 Fw for qRT-PCR OsARF19 Rv for qRT-PCR OsMADS62 Fw for qRT-PCR OsMADS62 Rv for qRT-PCR LOC_Os01g14440 Fw for qRT-PCR LOC_Os01g14440 Rv for qRT-PCR LOC_Os01g64360 Fw for qRT-PCR LOC_Os01g64360 Rv for qRT-PCR LOC_Os04g31730 Fw for qRT-PCR LOC_Os04g31730 Rv for qRT-PCR LOC_Os07g41580 Fw for qRT-PCR LOC_Os07g41580 Rv for qRT-PCR LOC_Os04g51000 Fw for qRT-PCR LOC_Os04g51000 Rv for qRT-PCR Oligo CRISPR OsFD4 Fw Oligo CRISPR OsFD4 Rv Oligo CRISPR OsFD1 Fw Oligo CRISPR OsFD1 Rv Oligo CRISPR OsFD3 Fw Oligo CRISPR OsFD3 Rv 

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Hepworth et al. International Journal for Equity in Health (2015) 14:51 DOI 10.1186/s12939-015-0183-x RESEARCH Open Access How an urban Aboriginal and Torres Strait Islander primary health care service improved access to mental health care Julie Hepworth1*, Deborah Askew1,2, Wendy Foley2, Deb Duthie3, Patricia Shuter1, Michelle Combo2 and Lesley-Ann Clements2 Abstract Introduction: Aboriginal and Torres Strait Islander people experience higher levels of psychological distress and mental ill health than their non-Indigenous counterparts, but underuse mental health services. Interventions are required to address the structural and functional access barriers that cause this underuse. In 2012, the Southern Queensland Centre of Excellence in Aboriginal and Torres Strait Islander Primary Health Care employed a psychologist and a social worker to integrate mental health care into its primary health care services. This research study examines the impact of this innovation. Methods: A mixed-method research design was used whereby a series of qualitative open-ended interviews were conducted with 7 psychology clients, 5 social work clients, the practice dietician, and the social worker and psychologist. General practitioners, practice nurses, Aboriginal Health Workers and receptionists participated in 4 focus groups. Key themes were identified, discussed, refined and agreed upon by the research team. Occasions of service by the psychologist and social worker were reviewed and quantitative data presented. Results: Clients and staff were overwhelmingly positive about the inclusion of a psychologist and a social worker as core members of a primary health care team. In one-year, the psychologist and social worker recorded 537 and 447 occasions of service respectively, and referrals to a psychologist, psychiatrist, mental health worker or counsellor increased from 17 % of mental health clients in 2010 to 51 % in 2012. Increased access by Aboriginal and Torres Strait Islander people to mental health care was related to three main themes: (1) Responsiveness to community needs; (2) Trusted relationships; and (3) Shared cultural background and understanding. The holistic nature and cultural safety of the primary health care service, its close proximity to where most people lived and the existing trusted relationships were identified as key factors in decreasing barriers to access. Conclusions: Improving social and emotional well-being is critical to addressing the health inequalities experienced by Aboriginal and Torres Strait Islander peoples. This study demonstrates the benefits for clients and health professionals of integrating culturally safe mental health services into primary health care. Keywords: Access, Cultural safety, Indigenous, Aboriginal and Torres Strait Islander, Mental health services, Primary health care, Integrated care, Qualitative research, Psychology, Social work * Correspondence: julie.hepworth@qut.edu.au 1 School of Public Health and Social Work, Queensland University of Technology, Brisbane, Australia Full list of author information is available at the end of the article © 2015 Hepworth 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. Hepworth et al. International Journal for Equity in Health (2015) 14:51 Introduction The continuing effects of colonisation, intergenerational trauma, and widespread social and economic disadvantage have contributed to the poorer health status of Aboriginal and Torres Strait Islander peoples compared to their non-Indigenous counterparts. Additionally, experiences of institutional racism have presented, and continue to present, a challenge in terms of engaging with health services, particularly mental health services [1, 2]. Ironically, these very issues also render Aboriginal and Torres Strait Islander peoples vulnerable to mental ill health [3]. Mental ill health has been estimated to make the second highest contribution (15 %) to disease burden in the Indigenous population accompanied by a disproportionally high level of unmet needs [2, 4] and further exacerbated by disproportionally low access to mental health services [5, 6]. Highlighting this gap, Fielke [7] observed; “Indigenous people do not enter mainstream care expecting to be treated well” (p.S75). As a result of reluctance to use mainstream mental health services, many Indigenous people do not receive early intervention and/or preventative care. Consequently, responses to Indigenous mental ill-health are frequently reactive and in response to crises, and at times may also involve the police [4, 8]. This situation results in a cascade of negative effects precipitated by misunderstanding and miscommunication – a negative experience for both staff and patients [4]. Such past failures, experiences of stigma and racism and a fear of being incarcerated perpetuate reluctance to access mainstream mental health care services within the community [4, 9]. The process of integrating mental health workers into Indigenous primary health care teams has been the subject of consideration by researchers and policy makers [7, 10–12]. Indigenous people have been shown to be more likely to visit an Indigenous mental health worker, and especially one who is highly visible in the community [7, 10]. Nagel [2] emphasises the imperative for mental health workers to focus on strengths based interventions and protective factors, with the objective of recovery for Indigenous clients. The National Aboriginal and Torres Strait Islander Health Plan: Key strategies for mental health and social and emotional well-being [13] acknowledges the holistic nature of social and emotional well-being (SEWB) and the need to support strategies that promote culturally safe services that integrate SEWB in all aspects of health care delivery for Indigenous communities. This research study examined the impact of integrating mental health care service delivery into an urban Aboriginal and Torres Strait Islander primary health care service. Page 2 of 8 Methods Aboriginal and Torres Strait Islander community approval and ethical clearance This study included a commitment to conducting research within an appropriate ethical framework as recommended by the National Health and Medical Research Council’s Values and Ethics – Guidelines for Ethical Conduct in Aboriginal and Torres Strait Islander Health Research [14]. Ethical research conduct involved two key processes. The Inala Community Jury for Aboriginal and Torres Strait Islander Health Research (a group of local Aboriginal and Torres Strait Islander people who guide all research undertaken by the Southern Queensland Centre of Excellence in Aboriginal and Torres Strait Islander Primary Health Care (Centre of Excellence) provided community support for the project [15]. Ethical clearance was obtained from the Metro South Human Research Ethics Committee. Results were disseminated back to the Inala Aboriginal and Torres Strait Islander community via the Community Jury at completion of the project and to the Centre of Excellence staff at a staff forum. Setting The Centre of Excellence (formerly known as the Inala Indigenous Health Service) is a Queensland Government general practice located in Inala, a South-Western suburb of Brisbane 18 km from the Brisbane central business district. In 2011 Aboriginal and Torres Strait Islander people made up 6.6 % of the population (910/ 13,796), significantly more than the national proportion of 2.5 % in the same year [16]. The Centre of Excellence has a long history of improving access to care, with an original practice population of 12 Aboriginal and Torres Strait Islander people in 1995 to approximately 10,000 adult patients in 2014, the majority of whom are Aboriginal and/or Torres Strait Islander who reside in Inala or surrounding suburbs [17]. In 2011, the Centre of Excellence received funding from the Queensland Government to initiate a program of activity aiming to improve the prevention, management and treatment of chronic disease. In recognition of the significant impact of chronic disease on mental health and SEWB, and the history of access barriers to culturally appropriate mental health care services, funding was allocated to employ a psychologist and social worker as core members of the primary health care team. In response to community feedback, we specifically targeted Aboriginal or Torres Strait Islander providers to fill each position. Study design The study was based on a mixed-method research design including two components: (1) descriptive statistics Hepworth et al. International Journal for Equity in Health (2015) 14:51 Page 3 of 8 on service access, and (2) a qualitative research approach [18]. We used a predominantly qualitative type of mixed-method design, a QUAL-quan [19], because of the paucity of existing research about the provision of mental health services for Aboriginal and Torres Strait Islander people in urban primary health care. This research design included the use of interview schedules with broad, open-ended question areas to inform a conversational style of data collection with both service clients and members of the primary health care team. Consequently, the identification of key themes in the interviews that reflected participants’ perspectives was achieved rather than pre-defined categories being imposed by the researchers onto the qualitative data. Given the large amount of qualitative data generated, this paper presents results about a specific area - the impact of the inclusion of a psychologist and social worker in a primary health care service on access to mental health services by Aboriginal and Torres Strait Islander people. The primary health care team participants included general practitioners (GPs), practice nurses (PNs), Aboriginal health workers (AHWs), receptionists, the dietician, the psychologist and the social worker. All team members were employees of the Centre of Excellence, eligible to participate, and actual participation was determined by availability at the times when the interviews occurred. Participants, sampling and recruitment The study included two groups of participants: (1) clients of the psychologist and/or the social worker, and (2) members of the primary health care team. Clients of the psychologist and social worker were eligible if neither of these two health care providers had provided active care within the 3 months prior to the research commencing and the health care providers considered they were socially and psychologically able to participate in the research. Purposive maximum variation sampling [18] was used to ensure inclusion of a range of clients seen by each health care provider (seven clients of the psychologist, five clients of the social worker, including two clients of both the psychologist and social worker), age (6–67 years, although the parent of the 6 year old was the interviewee), gender (four men, eight women), and referral type, referral condition and the number of appointments (14 appointments (n = 1), six appointments (n = 2), five appointments (n = 1), four appointments (n = 1), and three appointments (n = 3). To minimise selection bias, lists of potential client participants were prepared by the social worker and the psychologist to cover each parameter, and then one of the non-clinical research team members selected potential participants in each category. Based on a recommendation by the Community Jury, the psychologist and social worker telephoned the selected clients, gauged their well-being, informed them about the research and gained verbal consent for a researcher to contact them about participation in the research. Clients were then contacted by a researcher to further discuss the research, and, if agreeable, to arrange a mutually convenient time for an interview. Data collection and analysis (1)Descriptive statistics on service access The quantitative data identify the total number of occasions of service of the social worker and the psychologist, the number of referrals to the psychologist being made by the GPs, and the number of general practice mental health care plans commenced. To determine changes in clinical practice, we compared the proportion of mental health clients being referred to a mental health worker, counsellor, psychologist or psychiatrist in the year preceding the employment of the psychologist and the social worker (2010) and in the year after their employment (2012). (2)Qualitative research An Aboriginal member of the research team who was not an employee of the Inala Indigenous Health Service conducted the client interviews where possible in order to protect clients’ privacy, and to ensure maintenance of clients’ cultural safety. When this was not possible, interviews were undertaken by a non-clinical member of the research team with experience of conducting qualitative research in this community. Interviews took place in the clients’ homes (n = 3), at the health service (n = 7) or via the telephone (n = 2). Participants were each thanked with a AUD$25 gift card. Each interview aimed to cover similar broad areas including questions about their perceptions of the work of the social worker and/or psychologist, experiences of accessing a social worker and/or psychologist prior to the employment of these health professionals at the Centre of Excellence, expectations and experiences of appointments with the social worker and/or the psychologist at the Centre of Excellence, and suggestions for changes or improvements to these service. Further recruitment of participants was not required after the completion of 12 interviews because data saturation was achieved. Four focus groups (GPs n = 10; PNs n = 6; AHWs n = 5; receptionists n = 2) and one interview with the dietician were held with members of the primary health care team with each interview lasting approximately 35 min. The focus groups and the dietician interview covered Hepworth et al. International Journal for Equity in Health (2015) 14:51 Page 4 of 8 expectations of services provided by the social worker and/or psychologist, the impact of having the social worker and the psychologist as core members of the primary health care team, past and current referral processes for mental health care, patient outcomes resulting from the increased access and availability of the social worker or psychologist, and suggestions for improvements. Interviews with the psychologist and social worker covered expectations of their role, referrals, working inter-professionally and with other agencies and suggestions for how the service could be shaped in the future. A non-clinical member of the research team facilitated the focus groups and conducted the interviews in an attempt to minimise observer bias. The interviews and focus groups were digitally audiorecorded with permission, transcribed by a transcription service, and data were checked and de-identified prior to analysis. The only transcript corrections required pertained to the use of Aboriginal English. Mainstream English speakers often misunderstand this dialect of English, used consistently in the Aboriginal and Torres Strait Islander community, and these misunderstandings were evident in the transcriptions. First, each interview was summarised by two members of the research team allowing for the independent identification of key themes and sub-themes that were subsequently discussed with all members of the research team over a series of four meetings. During these discussions about the analysis, the key themes were compared across all interviews until the major and sub-themes were agreed upon by the research team. An important part of the analysis was the involvement of the social worker and psychologist who provided clarification about their roles and responsibilities and increased the accuracy of reporting the relationship between the themes and service delivery. Illustrative extracts from each of the themes were selected by the research team and are presented in the next section to demonstrate key issues voiced by participants and to ensure a range of perspectives. The results about access are reported in this paper. 2010, 17 % of mental health clients were referred to a psychologist, psychiatrist, mental health worker or counsellor external to the service, increasing to 51 % of mental health clients in 2012, after the psychologist and social worker were employed at the service. Results and discussion (1)Descriptive statistics on service access In the first 12 months that the social worker and psychologist were employed by the Centre of Excellence they recorded 447 and 537 occasions of service, respectively. GPs referred 226 clients to the psychologist, and 199 mental health care plans were commenced. Referrals to the social worker were not recorded in the practice management software as she did not have a Medicare provider number at the time of this study and therefore this information was not available for this research. In (2)Qualitative research The qualitative data demonstrates three key themes about Aboriginal and Torres Strait Islander people’s access to, and health service staff experiences of, mental health care services within a primary health care service. The three themes are: (1) Responsiveness to community needs; (2) Trusted relationships; and (3) Shared cultural background and understanding. Theme 1. Responsiveness to community needs A key way in which clients and staff talked about receipt of mental health care at the Centre of Excellence was to contrast the service with mainstream mental health services. Several problems that clients and staff had previously faced are described as well as what had improved and why since the inclusion of the psychologist and social worker within the Centre of Excellence primary health care team. All participants reported improved access and dramatic changes in the nature of mental health care that was now available through the Centre of Excellence. Mental health care at the Centre of Excellence versus mainstream services Prior to the employment of the psychologist and social worker at the Centre of Excellence, there were several access barriers for service users. In particular, although publicly funded mainstream services were available, clients had to travel approximately 16kms to access them. Both clients and health professionals identified that local services were needed in order to remove the significant logistical and attitudinal obstacle of having to travel away from their secure environment, especially at times of crises. If I had to travel across town, I probably wouldn’t have done it. That would have just been [impossible] – well, at that point in time (Woman, 53 years old). Also, in using the mainstream system there was no cultural connection and service users shared the view that seeing an Indigenous health care provider was important. I was seeing other psychologists outside the services and I went through about 10 of them. Didn’t click. So I thought it might have been a cultural difference (Woman, 43 years old). Hepworth et al. International Journal for Equity in Health (2015) 14:51 Page 5 of 8 Similarly, health service staff described mental health services in Queensland as generally being a negative experience for Aboriginal and/or Torres Strait Islander peoples because of the geographic, bureaucratic and cultural barriers to accessing appointments, a lack of understanding about Indigenous culture and SEWB especially that much of the work involved service users presenting in crises - and the more general limitations of the mainstream mental health system, such as a lack of communication and integration among health care providers. social worker. However, these referrals tended to occur without communication and a lack of awareness about existing client load or local practice in treatment plan development. Consequently, these agencies have required education about the roles and clinical processes of the social worker and psychologist, and clarification of the differences between their therapeutic roles and the roles of the referring agencies: I think mental health in Queensland is poorly done. Access to services for Aboriginal people is pretty pathetic (GP: Focus group). There have definitely been lots of individual reports from people about how they might have been referred to psychological services … in the past but never actually went for reasons: comfort talking to another Indigenous person; no local clinic; payment … stigma and misunderstanding (Psychologist: Interview). Holistic, culturally appropriate care In contrast to mainstream mental health services, staff described inclusion of mental health services within the primary health care team at the Centre of Excellence as enabling the provision of “holistic” and “culturally appropriate” care, increasing accessibility, and having a positive impact on service users. [The Centre of Excellence provides] holistic care. There’s one young fellow came in quite disturbed and then I referred him to the social worker and then just seeing him about a month down the track, smile on his face. And so the system does work and I just was thrilled about holistic care because I’ve worked in a lot of places and all (PN: Focus group). I say to [patients] ‘[The agencies] can refer you to our service and they can refer you to our clinic or my name on that plan but not actually tell me or ask me … but once you come here, how often we meet and what we talk about is up to us’. If [the agencies] tell someone they’ve got to come weekly I will decide whether I need to see them weekly or not and I give [the agencies] that feedback and they often accept it (Psychologist: Interview). The contrast between mainstream (or external) mental health services and the Centre of Excellence is very pronounced whereby the former is reported to have multiple access barriers for Aboriginal and Torres Strait Islander people. This finding is consistent with existing research that has identified a range of challenges for Indigenous peoples accessing mental health services [1, 2], and in turn the lack of appropriate services contributes to worsening mental ill health [3]. The integration of a psychologist and social worker within the Centre of Excellence in ways that were responsive to the needs of Aboriginal and Torres Strait Islander people created mental health services that were delivered differently. This was based in part on the shared understanding that what was required of the service was distinctly different from mainstream services and would remain negotiated and responsive to service users. Theme 2. Trusted relationships They do a lot better than – in my opinion, than the mental health mainstream system does because they’re easily accessible and they are culturally appropriate. The turnover is quite good. I think within a week you can see [someone] (AHW: Focus group). There was a different type of atmosphere working with our community, with your own community. Like I know a lot of clients from a family perspective … and still have that professional boundaries (Social Worker: Interview). An unexpected consequence of the inclusion of the mental health services into the core business of the Centre of Excellence was referrals by other agencies, particularly government agencies, of Aboriginal and Torres Strait Islander people to the psychologist or Service users described their experience of accessing mental health care services at the Centre of Excellence in terms of the importance of supportive relationships and feeling “comfortable” in the environment. The whole Inala [Indigenous] Health [Service], Murri Health, they’re so approachable, and the nurses. Somebody should do something big for them. They’re just beautiful, comfortable, and it’s so caring that it’s not – I can’t think of the word for that, not feel sorry for you (Woman, 43 years old). Health service staff talked enthusiastically about the strong relationships that underpinned the care provided by the Centre of Excellence, and attributed increased access to mental health services to several relational Hepworth et al. International Journal for Equity in Health (2015) 14:51 Page 6 of 8 features. Importantly, patients of the Centre of Excellence were already familiar with the longstanding Aboriginal and Torres Strait Islander primary health care service and they had existing positive and trusting relationships with the health service staff that enabled greater ease of access. The clinic staff regularly referred to these trusting relationships as the following extracts illustrate: asked about potential stigma resulting from seeing a psychologist they did not see clients as being concerned. There’d be elements of trust, where they trust the psychologist because they’re under our roof – I think there’d be elements of just the access where they know where it is and they know how to get there and they’re comfortable in that surroundings (GP: Focus group). It comforts the patient too to know that that’s a one-stop shop and they don’t have to go outside, because so many patients feel comfortable in our service (Dietician: Interview). The staff explained how the dynamics of the relationships between health service staff and service users was pivotal to ongoing care and these relationships facilitated and promoted the provision of effective health care. Also, [the social worker and psychologist are] quite – they’re full-time so it’s not something [clients] have to wait around for either, so it’s – they get in quite quickly and they can build a relationship because they’re there all the time, so they don’t have to go from one to the other and then, I guess, the thing is when people have to travel outside of Inala, the chance of them going isn’t always quite high (AHW: Focus group). More freely [refer] – often there’s lots of issues. People never come in with one – with their sore foot or whatever. They might have the sore foot but there’s all the other stuff that goes on. So you could actually talk about that and say that we have these services but if you’re thinking, trying to refer that externally that just wouldn’t work (PN: Focus group). While previously referrals had been made to external mental health services these had not worked well, and in many instances clients had simply not attended. The psychologist gave an example of a conversation she had had with a male client who she and the community considered to be an Elder who had told her that he and those he knew would not attend anywhere else. She stated that his message was clear; “If you don’t continue don’t refer us to anyone because we are not going to go”. The fact that clients were comfortable using the service was also recognised by the reception staff, and when the staff were I think they appreciate it, being so close and not having to travel … I don’t think – no, I don’t think anyone’s worried about going to see the psychologist (Receptionist: Focus group). Here, the participants have illustrated the relational dynamics involved in building trust and its relationship with increasing access. As Fielke et al. [7] argue, Indigenous people do not expect to be treated well entering services, which in turn, highlights the ways in which the relational aspects of the mental health care at the Centre of Excellence functioned to enable and maintain access sufficiently for clients to obtain advice and support. Theme 3. Shared cultural background and understanding The significance of shared cultural understanding was important for the clients. As mentioned above, the psychologist and social worker were both Aboriginal, and therefore clients felt a sense of comfort and familiarity that contributed to the development of a therapeutic relationship. This was particularly important given the feelings of shame associated with mental health issues in the community. Shared cultural identity From the perspectives of service users being able to identify with a health care provider who was Aboriginal and/or Torres Strait Islander had positively contributed to improving service access and its effectiveness. In both extracts below, provided by a woman and a man, it was the experience of the social worker and the psychologist being Aboriginal that had enabled the clients to obtain help because of the shared understanding of the similar issues that they faced. I think it helped that she [the psychologist] was a black woman … young and Aboriginal … because we have this – deal with the same issues and stuff. I don’t know her background and stuff but as black women, we get dealt with the same stuff in everyday society (Woman, 29 years old). She knew where we were coming from, ‘cause she’s an Aboriginal lady too, like – so she knew half the problems and that and where I was coming from and even with the kids and stuff like that (Man, 49 years old). The cultural significance of the psychologist and social worker having Aboriginal backgrounds cannot be overestimated. In the extracts directly below, as in other Hepworth et al. International Journal for Equity in Health (2015) 14:51 Page 7 of 8 extracts presented in the analysis under related themes, the connectedness of service users and health service providers through their Aboriginality was clearly beneficial to accessing the service and its therapeutic effectiveness. space both service users and staff recognised that its location contributed to privacy. Having Aboriginal and Torres Strait Islander staff outstandingly builds trust – there is a risk for mainstream services to not really understand the barriers or the difficulties that people face in their life – they think they are going to get judged … and so it has outstandingly overcome those barriers of access (GP: Focus group). Often they’ll know a relative of the social worker so, straight away, they feel that connection and they’re happy to go – because, definitely having somebody of Aboriginal background in the social worker or psychology role is, I think in the service, really crucial, so that’s often that turning point for people in that decision-making process (Dietician: Interview). Although, having a shared cultural background was not unproblematic. Other health care staff discussed very different views about a shared cultural background in terms of the fears held by some service users that their mental health issues may become known within their own communities. I’ve got a girl at the moment who’s got postnatal depression and she said that she didn’t want to know the psychologist. “I don’t want – I’m Murri [an Aboriginal person from Queensland]. I’m Murri”. She didn’t want a Murri, because that just conflicts with her and the family members might find out (Nurse: Focus group). Here, the concern held by some service users that their privacy might be compromised if they consulted an Aboriginal health service provider is clear. Aboriginal peoples value an extended family system, and family is an integral part of an Aboriginal person’s life. These extensive networks can contribute to concerns about a lack of privacy and sharing of personal information which could mean that family members become aware of mental health problems. Shame and mental health Health service users also identified a fear that other community members might find out about another family member’s mental health and/or social problems. Even though the decision to physically locate the psychologist and social worker in a different building from the general practice clinic was due to a lack of available space in the clinic and not intended to provide a more private It was in another part of the building which – I guess again, you know, we’ve always got those issues around, you know, we don’t want people blabbing about our business to all them other fellas, and I sort of felt that I could trust her that way (Woman, 53 years old). I was a bit sceptic at the start because Aboriginal people being – like, everyone knows someone of someone. … Yeah, once I’d met ‘em spoke to ‘em, I was really, really at ease, yeah (Man, 32 years old). Sometimes I felt like people knew if you were going to counselling, and when you’re down and out like that it’s big shame. Yeah, I don’t feel shamed now but when you’re down and you do feel that, at that time. At first I thought it would be that way, but she just greeted me (Woman, 43 years old). The fear of being identified by their families and community was strongly linked to a feeling of shame. Health service staff identified shame as being a common concern for service users, and the AHWs’ and social worker’s excerpts below illustrate the significance that feelings of shame and the need for privacy have for service users. It’s probably shameful for any Murri person to sit there and access a service, so the fact that they’re located just around the corner, it’s isolated and there’s a few different staff members that sit there, so they don’t always know that it’s about mental health issues (AHW: Focus group). And it’s trust too because the families are very big and they don’t want everyone to know that they’ve been here to see you (Social Worker: Interview). In the extracts above the importance of privacy is clearly highlighted, but once the service had been accessed a lot of fears were allayed. The availability of a range of Aboriginal and/or Torres Strait Islander health care providers at the Centre of Excellence has contributed to improvements in access for Aboriginal and Torres Strait Islander people to comprehensive primary health care. This is consistent with research that has demonstrated that a shared cultural identity enhances attendance at appointments [7, 10]. Given that shame is such a pervasive issue related to mental health [4, 9], one of the significant achievements of the Centre of Excellence was to sufficiently engage clients to access the service and experience a culturally safe environment. Hepworth et al. International Journal for Equity in Health (2015) 14:51 The three themes of responsiveness to community needs, trusted relationships and shared cultural identity, represent key features of the development and delivery of health care for social and emotional well-being within a framework of cultural safety. Conclusion The experiences of health service users and staff of the addition of a psychologist and social worker to the primary health care service were overwhelmingly positive and access was significantly improved. Responsiveness to the needs of the Aboriginal and Torres Strait Islander community in the design, development and delivery of health care, trusted relationships and a shared cultural understanding were main features that contributed to the success of the expanded primary health care service. Improving social and emotional well-being is crucial to addressing the health inequalities experienced by Aboriginal and Torres Strait people. This study demonstrates the benefits of integrating culturally safe mental health services into primary health care. Competing interests This research was supported in part by a small consultancy from Queensland Health that provided some research assistance by PS, the initial analysis by JH, and transcription of the interviews. Authors’ contributions JH contributed to the conceptual design, made a significant contribution to the analysis and drafted the manuscript. DA contributed to the conceptual design, and contributed to the data analysis and manuscript. WF contributed to the conceptual design, developed the ethics application, selected participants, conducted a focus group and patient interviews, worked on quantitative data, checked all transcripts and contributed to the analysis and manuscript. DD contributed to the data collection, analysis, and manuscript. PS contributed to the literature review, data collection and analysis. MC contributed to conceptual design, provided patient list, contributed to analysis and manuscript. L-AC contributed to the conceptual design and analysis. All authors read and approved the final manuscript. Acknowledgements We acknowledge the contribution of members of the local Aboriginal and Torres Strait Islander community to this research, and thank them for trusting us with their stories. We also acknowledge the staff of the Centre of Excellence for their contributions to this research. Author details 1 School of Public Health and Social Work, Queensland University of Technology, Brisbane, Australia. 2Southern Queensland Centre of Excellence in Aboriginal and Torres Strait Islander Primary Health Care, Brisbane, Australia. 3Oodgeroo Indigenous Support Unit, Queensland University of Technology, Brisbane, Australia. Received: 3 December 2014 Accepted: 1 May 2015 Page 8 of 8 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. Vicary D, Bishop B. 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Preparation and nutrient release kinetics of enriched biochar-based NPK fertilizers and agronomic effectiveness in direct seeded rice Arkaprava Roy Govind Ballabh Pant University of Agriculture & Technology Sumit Chaturvedi (  sumitagronomy78@gmail.com ) Govind Ballabh Pant University of Agriculture & Technology Suhita Pyne Govind Ballabh Pant University of Agriculture & Technology Shiv Vendra Singh Govind Ballabh Pant University of Agriculture & Technology Govindaraju Kasivelu Sathyabama Institute of Science and Technology Research Article Keywords: Slow release, biochar, conventional fertilizers, release pattern, nutrient use efficiency Posted Date: September 15th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-904406/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/16 Abstract In present study, two enriched biochar-based fertilizers were prepared having fertilizer grade of 6-6-4 N-P2O5-K2O by intercalation of NPK fertilizers mixture solution as EB-1 and additional humic acid and seaweed extract as EB-2. In laboratory, batch experiment were done to compare nutrients (NH4+, NO3−, P and K+) release patterns of developed fertilizers along with conventional fertilizers. Enriched biochar fertilizers (EB) demonstrated much slower release pattern of NH4+, P and K+, however NO3− release was similar over conventional fertilizers. The cumulative release of N in EB fertilizers was similar to conventional fertilizer, however significantly less of P and K were released during the period of 72 hrs. The field response study of enriched fertilizers EB-2 revealed 29.5, 11.5 and 22.9% higher apparent use efficiency than conventional fertilizer. The slow nutrients release behaviour of EB fertilizers implies reduced losses and enhanced NUE as reflected by higher apparent recovery of N, P and K. Introduction Nutrient use efficiency (NUE) is a vital concept for assessing farming system which is greatly dependent on fertilizer management as well as soil- plant-water relationships. The nutrient use is mainly used to increase the crop performance by providing optimum nourishment while minimizing nutrient losses from the field and supporting sustainability by contributing to soil quality components. Use efficiency of nitrogenous fertilizers is around 30-40 %, phosphate fertilizers are 15-20 %, potassic fertilizers is 50-60 % and micronutrient is 1-2 % (Singh et al. 2014). Among the different approaches initiated to increase the nutrient use efficiency; control release fertilizer may be a novel environment-friendly approach. Control-release fertilizers delays the availability of nutrients after application for plant uptake and use, or which extends the nutrient availability to the plant for significantly longer period compared to the conventional fertilizers and thereby enhance nutrient use efficiency. Biochar prepared from agricultural wastes viz., rice straw and husk may be utilized as a suitable matrix to prepare control release fertilizers (Singh et al. 2020; Chaturvedi et al. 2021). It will not only enhance NUE but also improve soil health and crop yield with most economic management of agro-wastes. Biochar is a product of the pyrolysis of organic materials such as wood, rice husk, rice straw, leaves, grasses, crop residues and manure as a stable, recalcitrant organic carbon-rich amendment to improve soil bio-physiological and chemical properties (Lehmann et al., 2006). Biochar has a porous structure, huge surface area, a large number of functional groups, and plentiful mineral elements, that benefit loading of fertilizer nutrients and availability of heavy metals in the soil (Dong et al. 2019; Liu et al. 2019). Presence of surface functional groups on biochar strongly adsorbs various nutrient ions, including NH4+, NO3-, P and K+ ions (Kimetu 2010; Mizuta et al. 2004). This promotes to load nutrients on biochar and reduce their losses (Singh et al. 2020; O'Connor et al. 2018). Sorption by the biochar is the primary reason for reduced mean cumulative leaching of nitrate and nitrite in biochar-amended soil (Mukherjee et al. 2014). Therefore, application of biochar as a soil amendment or slow-release fertilizer could make farming more productive by compensating for acidity, improving organic carbon, water retention and nutrient availability and bring more area under agriculture (Fryda and Visser 2015). Despite the positive impacts of biochar in agricultural systems, it is usually very expensive to apply mainly due to the high cost of collecting feedstock as well as the high costs of pyrolysis plants. Biochar itself does not contain enough nutrients for crop growth (Yaung et al. 2016). To utilize biochar in agriculture comfortably, it can be enriched with chemical fertilizers. This can curtail cost and make it relative to that of conventional chemical fertilizers. Compared to conventional chemical fertilizers, biochar enriched fertilizers have been shown to increase crop yields, nutrient use efficiency, plenty of beneficial microorganisms, and lower soil greenhouse gas emissions (Joseph et al. 2013; Qian et al. 2014; Zheng et al. 2017; Yao et al. 2015; Blackwell et al. 2015). Sole application of biochar reduced grain yield (Asai Page 2/16 et al. 2009) due to the insufficient supply of nitrogen thus, augmenting biochar with fertilizer nutrients makes biochar composite suitable for plant growth and yield (Si et al. 2018) and heavy metal adsorption (Zhao et al. 2016). Due to the farming and ecological applications, enriched biochar fertilizers have received growing attention. Recent studies suggest that biochar-based fertilizers delay the release of nutrients in the soil displaying a slow-release effect (Chaturvedi et al. 2021). Enrichment of soil with biochar-based fertilizer adjust the soil pH, reduce the bulk density, improve soil aeration and water permeability and retention to increase the crops yield (Gao et al. 2012; O'Connor et al. 2018). Addition of enriched biochar in soil as a carrier of nutrients is enormous. Moreover, it could guide nutrient release in sustained manner and extended period due to its micro porous structure and extensive surface area (Ghezzehei et al. 2014; Singh et al. 2020). Collectively, these characteristics makes biochar-based fertilizer a novel approach that could reduce losses, increase nutrient bioavailability, mitigate GHG emission, improve soil health and consequently crop growth and biomass yield. Materials And Methods Synthesis and chemical characterization of biochar and enriched biochar-based fertilizers Synthesis of the biochar-based NPK fertilizer entailed three consecutive steps: (1) pyrolysis of rice husk for the production of the biochar for fertilizer carrier; (2) impregnation of the biochar with nutrient solution; and (3) granulation or prilling. The rice husk, a readily available low cost biowaste was obtained from Norman E. Borlaug Crop Research Centre, G.B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand (28.97°N, 79.41°E). Pyrolysis of prepared feedstock was carried out in rotary type reactor under oxygen-limited conditions at temperature of 450oC (Mandal et al., 2018), aiming to produce small pores and preserve oxygen-functional groups (Al-Wabel et al., 2013; Keiluweit et al., 2010; Lawrinenko and Laird, 2015; Yuan et al., 2014). An NPK nutrient solution was prepared by dissolving 12-32-16 NPK (IFFCO) + Urea + MOP mixture in distilled water. Prepared biochar was ground in disc mill using 60 mesh sieves after grinding and added to prepared NPK solution (NPK (12-32-16) fertilizer (200 kg) + Urea (80 kg) + MOP (20 kg) in ratio 7:3 to produce enriched biochar; EB-1 and additionally humic acid and seaweed extract was added for enriched biochar; EB-2. Both EB-1 and EB-2 had 6-6-4 N-P2O5-K2O fertilizer grade. The developed fertilizers were characterized for their properties and nutrient release kinetics. Determination of nutrient release patterns Batch experiment was conducted IFS laboratory with soil sample, Conventional fertilizer mix (N-P-K 12-32-16 + Urea + MOP), EB-1 and EB-2. Leachates of nutrient enriched biochar were collected through batch experiment for 36 h at 0 min, 15 min, 30 min, 1 h, 3 h, 6 h, 9 h, 15 h, 24 h and 36 h and NPK content in those leachates were determined as ammonium (NH4+), nitrate (NO3-), phosphorus (P) and potassium (K). Ammonium (NH4+) For determination of release pattern of NH4+ from conventional fertilizers mix, EB-1 and EB-2 batch experiment was followed. These three fertilizers were taken in 10 conical flasks for each sample on 1g N content basis. After that 10 g of soil sample from experimental field was added to each flask and 30 mL of water also added to each flask. At predetermined time intervals selected flasks samples were filtrated in 100 mL volumetric flask and after collecting filtrate filled the volumetric flask up to 100 mL. After collecting all the filtrates at all predetermined time intervals. Modified kjeldhal method was followed to determine NH4+ content in samples. Calculated NH4+ content (ppm) in samples from recorded readings were plotted in graph against time intervals to get NH4+ released pattern of these three fertilizers. Both the cumulative and instantaneous concentration graph at predetermined time intervals were drawn. Page 3/16 Nitrate (NO3-) After determination of NH4+ content of the samples, 0.2 g of Devarda’s alloy was added to the samples and again Modified kjeldhal method was followed to determine NO3- content in samples. Calculated NO3- content (ppm) in samples from recorded readings were plotted in graph against time intervals to get NO3- released pattern of these three fertilizers. Both the cumulative and instantaneous concentration graph at predetermined time intervals were drawn. Phosphorus (P) For determination of release pattern of P from conventional fertilizers mix, EB-1 and EB-2 batch experiment was followed. These three fertilizers were taken in 10 conical flasks for each sample on 0.1 g P2O5 content basis. After that 10 g of soil sample from experimental field was added to each flask and 30ml of water also added to each flask. At predetermined time intervals selected flasks samples were filtrated in 100 mL volumetric flask and after collecting filtrate filled the volumetric flask up to 100 mL. After collecting all the filtrates at all predetermined time intervals, ammonium molybdate and potassium antimony react method was followed to determine P content in samples. Calculated P content (ppm) in samples from recorded readings were plotted in graph against time intervals to get P released pattern of these three fertilizers. Both the cumulative and instantaneous concentration graph at predetermined time intervals were drawn. Potassium (K) For determination of release pattern of K from conventional fertilizers mix, EB-1 and EB-2 batch experiment was followed. These three fertilizers were taken in 10 conical flasks for each sample on 0.1 g K2O content basis. After that 10 g of soil sample from experimental field was added to each flask and 30 mL of water also added to each flask. At predetermined time intervals selected flasks samples were filtrated in 100 mL volumetric flask and after collecting filtrate filled the volumetric flask up to 100 mL. After collecting all the filtrates at all predetermined time intervals, Flame emission spectrophotometry method was followed to determine K+ content in samples. Calculated K content (ppm) in samples from recorded readings were plotted in graph against time intervals to get K released pattern of these three fertilizers. Both the cumulative and instantaneous concentration graph at predetermined time intervals were drawn. Evaluation of the Nutrient-use efficiency A field experiment was initiated during rainy season of 2018 at N.E. Borlaug Crop Research Centre, G. B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand to evaluate the effect of enriched biochar-based fertilizers on nutrient-use efficiency in direct seeded rice (DSR). Rice variety “HKR-47” was used for present study. The soil of experimental site was sandy loam in texture with bulk density of 1.44 Mg/m3. The soil was neutral (pH 7.2) in reaction, medium in soil organic carbon (0.65 %), low in available nitrogen (149.6 kg/ha), high in available phosphorus (26.9 kg/ha) and potassium (281.1 kg/ha). The moisture content of the site at field capacity and permanent wilting point was 20 and 8 %, respectively. The experiment had eight treatments applied at 3 rates with 3 fertilizer source i.e. 100 % RDF (120 kg N, 60 kg P2O5, 40 kg K2O / ha) through conventional fertilizers (T1); 75% (T2), 100% (T3) and 125% (T4) RDF through enriched biochar based fertilizer EB-1 for basal application + urea enriched biochar (UEB-1) containing biochar + urea (1:1) for top dressing of N; 75 % (T5), 100 % (T6) and 125 % (T7) RDF through enriched biochar based fertilizer EB-2 for basal application + urea enriched biochar (UEB-2) containing biochar + urea (1:1) + seaweed + humic acid for top dressing and no fertilizer application (T8) replicated thrice laid out in randomized block design. The recommended fertilizer dose (RDF) was 120 kg N + 60 kg P2O5 + 40 kg K2O per ha. half dose of N and full dose of P and K were applied through Page 4/16 conventional fertilizers mix as basal and rest half of N was applied in two equal splits at 30 and 60 DAS as top dressing through urea for treatment T1. Whereas for treatments T2, T3 and T4, half dose of N and full dose of P and K were applied through EB-1 as basal and rest half of N was applied in two equal splits at 30 and 60 DAS as top dressing through UEB-1. For treatments T5, T6 and T7, half dose of N and full dose of P and K were applied through EB-2 as basal and rest half of N was applied in two equal splits at 30 and 60 DAS through UEB-2 as top dressing. For evaluating nutrient-use efficiency of enriched biochar-based fertilizers apparent nutrient recovery (%) was determined using following formulae: Results And Discussion Characteristics of biochar and enriched biochar (EB-1 and EB-2) The surface morphology of biochar derived from rice husk is primarily performed by SEM and showed a highly porous structure with smooth and tight. The porous tubular morphology of rice husk biochar (RHB) favour it to store/reserve the nutrients and slow release to soil system (Fig. 1a&b). The properties of rice husk biochar (RHB) and EB-1 & 2 prepared are shown in Table 1. The RHB had pH value of 9.1, cation exchange capacity of 27 cmol/kg, EC of 1.55 ds/m and bulk density of 0.22 g/cc whereas, EB-1 and EB-2 had pH of 8.07 and 7.6 and EC of 0.15 and 0.17 ds/m and CEC of 17 and 21 cmol/kg, respectively. Proximate and elemental analysis representing the recalcitrance and stability of biochar relates the qualitative traits of resultant biochar (Table 1). These results are common for biochar from different feedstock’s produced at low temperature (Chaturvedi et al., 2021; Shaheen et al., 2019). Kinetics of NPK release Ammonium (NH4+) release pattern Ammonium (NH4+) release from conventional fertilizers mix (NPK 12-32-16 + Urea + MOP) reached its peak around 30 min after starting the batch experiment, whereas the peak NH4+ releases from both the enriched biochar fertilizers (EB-1 and EB-2) were noticed around 6 h after starting the batch experiment (Fig.2a&b). Ammonium (NH4+) released from conventional fertilizers mix at significantly faster rate compared to both of the enriched biochar based fertilizers. From the conventional fertilizers mix around 78.90 % of NH4+ released within 1 h but that same amounts of NH4+ (81.11 % and 81.78 %) were released from both of EB-1 and EB-2 respectively at 24 h after starting the experiment. This slow release of NH4+ might be due to the fact that hydrogen bonding and electrostatic interactions between the oxygen containing functional group (carboxyl and keto group) of biochar and NH4+, slowed the release rate of NH4+ from enriched biochar based fertilizers (Wen et al., 2017). Under pyrolysis temperature of 200 to 400 ℃, the feedstock was carbonized incompletely, so numerous oxygen functional groups were remaining in biochar, which could provide abundant negative potential charges for the adsorption of NH4+, owing to hydrogen bonding and electrostatic Page 5/16 interactions (Cai et al., 2016). In the present study, NH4+ released from the conventional fertilizers mix at the logarithmic rate i.e. released at faster rate with shorter time interval up to 1 h, after that release rate with increase in time interval were less and at the end of the experiment (36 h) around 96 % of NH4+ was released. In case of NH4+ releases from the EB-1 and EB-2, initial release rates were at par with conventional fertilizers mix but after that NH4+ released at slow and steady rate with time. Between the times interval 1 h to 15 h both the enriched biochar based fertilizers released around 54-55 % of total NH4+. At the end of the experiment (36 h) total NH4+ released from both of the enriched biochar fertilizers were at par with conventional fertilizers mix. There was no significant difference in NH4+ release pattern for both of EB-1 and EB-2. Similarly, rapid release of urea was recorded with RHB-450 and most of the urea released for 2900 min and thereafter get plateau whereas in case RSB-450, increasing release trend was recorded upto 3600 min (Singh et al., 2020). Liu et al (2019) reported release from the urea loaded biochar and bentonite composites to follow similar trend. Swelling and spelling of biochar with the absorption of moisture lead to the release nutrient particles intercalated nutrient particles into the cavities of biochar. Nitrate (NO3-) release pattern Nitrate (NO3-) nitrogen released from both EB-1 and EB-2 was more as compared to the conventional fertilizers mix (Fig. 3a&b). It was due to less nitrate content in conventional fertilizers mix because of this conventional fertilizers mix contented most of the nitrogen in ammoniacal (NH4+) form. Though enriched biochar based fertilizers were enriched with that same fertilizers composition, there might be chance to convert NH4+ to NO3- due to reaction with biochar matrix. There was no significant difference in NO3- release pattern for EB-1 and EB-2. Similar release pattern was reported by Gwenzi et al (2017) for biochar based slow-release fertilizer (BSRF). Phosphorus (P) release pattern In general, enriched biochar based fertilizers (EB-1 and EB-2) had consistently lower phosphorus release than the conventional fertilizers mix throughout the 36 hours monitoring period (Fig. 4a&b). Total phosphorus release was significantly lower for EB-1 and EB-2 than conventional fertilizers mix up to 9 h of starting of batch experiment and after that total releases were statistically at par. P release pattern for conventional fertilizers mix was characterized by general decline with time with its peak at 3 h after starting the experiment. The peak of P release pattern for both of the enriched biochar fertilizers were different. P release from EB-1 reached its peak around 6 h and from EB-2 reached its peak around 9 h after starting the experiment. Gwenzi et al. (2017) also found that in P release pattern the cumulative concentration of PO43- released from biochar based slow release fertilizer (BSRF) was approximately half of that of commercial fertilizer Compound D during a 72 days’ period of sequential leaching. In the present study, there was no significant difference in P release pattern for both of EB-1 and EB-2. The P in the matrix of biochar is slowly released due to physical protection of the biochar pores network (Dias et al., 2018). The water action removes aliphatic groups (hydrophobicity) on biochar surface, increasing its affinity for water (Das and Sarmah, 2015) interacting with hydrophilic groups resulting a swelling of the biochar coating making a new arrangement which finally controls the water movement inside and outside of the fertilizer (Dias et al., 2018). Till date, limited studies have evaluated nutrient release patterns of slow release NPK fertilizer using biochar as a nutrient carrier. Yao et al. (2013) used biochar to remove P from aqueous solution and concluded that P-laden biochar could possibly act as a slow-release fertilizer. It did not include other nutrients such as cations and highly soluble and mobile nutrients in its study. Moreover, a conventional chemical fertilizer was also not taken for comparison. Therefore, the present study focused to develop a biochar-based slow-release NPK fertilizer and evaluate its nutrient release pattern. Potassium (K+) release pattern Page 6/16 Peak K+ releases from the conventional fertilizers mix as well as from the enriched biochar based fertilizers (EB-1 and EB-2) occurred at 1 h after starting the experiment but that for EB-1 was about 60.50 % and for EB-2 was about 44.22 % lower than that of conventional fertilizers mix (Fig. 5a&b). Overall cumulative concentration of K+ released by both EB-1 and EB-2 were lower than that for conventional fertilizers mix up to 36 h of experiment. Similar result was reported by Gwenzi et al. (2017) that overall cumulative concentration of K+ released by biochar based slow release fertilizer (BSRF) was 1.5 times lower than that of for commercial fertilizer Compound D and peak K+ release was significantly lower for BSRF than that of Compound D. In the present study, there was no significant difference in K+ release pattern for both of EB-1 and EB-2. Evaluation of NH4+, NO3-, PO43-, and K release patterns confirmed the slow-release behavior of the biochar enriched fertilizers (Figs. 2-5). Nutrient release patterns of biochar enriched slow-release fertilizers were characterized by an initial high release in the first few observations mainly attributed to rapid diffusion and washout as also reported by Gwenzi et al. (2017). The release of PO43- and K by enriched fertilizers was characterized by distinct multiple peaks suggesting a number of release mechanisms which was not the case with N. Moreover, the cumulative PO43- and K releases of the enriched fertilizers (EB-1 &EB-2) were approximately half that of commercial fertilizer mix in period of study. The manifold mechanisms of nutrient release from biochar enriched fertilizers include pore diffusion, ion exchange on charged biochar surfaces of biochars, co-precipitation, complexation/ligand exchange, van der Waals/electrostatic forces, and p-cation interactions between solute ions and aromatic rings of biochar (Wang and Chen 2006; Berber-Mendoza et al., 2013). Moreover, the use of an adjuvants may enhance the slow-release behavior of the biochar-based fertilizers. Apparent nutrient recovery (%) of N, P and K Apparent nitrogen recovery was higher under application of 100 % fertility dose through EB-2 + UEB-2 which was 29.5 % more than application of conventional fertilizers at that same fertility dose (Table-2). Even application of EB-1 + UEB-1 at 75, 100 and 125 % fertility levels had lower apparent N recovery as compared to EB-2 + UEB-2 at similar fertility levels, respectively. However, application of both the enriched biochar fertilizers at all the fertility levels had more apparent N recovery as compared to application of 100 % fertility level through conventional fertilizers. More N uptake from enriched biochar sources might be the principle reason for their higher apparent recovery. Apparent P and K recovery was higher under application of 100 % fertility dose through EB-2 + UEB-2 of 29.39 % and 83.06 % of P and K respectively. Further, it was 11.5 % and 22.9 % more than application of conventional fertilizers at that same fertility dose (Table-2). Huang et al. (2013) reported that due to addition of biochar, agronomic nitrogen use efficiency was increased by 43 % over no biochar addition at 100 % fertility level. Slow release of nitrogen, phosphorus and potassium from NPK enriched biochar-based fertilizers is mainly attributed to their slower release kinetics, reduced losses and indirectly by enhanced availability of soil nutrients thereby higher apparent use efficiency of N, P and K under enriched biochar fertilizers application. Several studies have shown that biochar application enhanced the microbial activity including soil enzyme activity and soil respiration (Steiner et al., 2008), promote root colonization by arbuscular mycorrhiza (Elmer and Pignatello, 2011). These root exudates and enhanced microbial activities could further promote release of nutrients from insoluble soil nutrient pools. Therefore, biochar based slow-release fertilizers have several potential niche applications in crop production, horticulture, olericulture including environmental remediation. Conclusions Two novel enriched biochar-based fertilizers (EB-1 and EB-2) were developed with much slower nutrient release patterns as compared to that of conventional chemical fertilizers. The slow nutrients (NH4+, P and K+) release behavior Page 7/16 of EB fertilizers implies reduced loss of nutrients through leaching and hence increase crop nutrient use efficiency. Chemical characterization of EB fertilizers showed their potential niche applications in agriculture including environmental remediation demonstrating their potential superiority over conventional fertilizers mix. Declarations Acknowledgement The author (SC) thank to ICAR-NICRA, CRIDA, Hyderabad, Government of India for financial support. Declaration of Competing Interest All the authors declare that they have no known competing financial interests. Compliance with ethical standards: Not applicable Funding: This study was supported by ICAR- National Innovations on Climate Resilient Agriculture (NICRA), Hyderabad, Government of India. Conflict of interest: All authors declare that they have no conflict of interest. Ethics approval/declarations: Not applicable Consent to participate: Not applicable Consent for publication: Not applicable Data Availability Statements: All the data generated or analysed during this study are included in this article Author contribution: Arkaprava Roy: data collection, analysis, Sumit Chaturvedi: writing, reviewing, and editing, Suhita Pyne: designing the experiments, analysis, Shiv Vendra Singh: analysis, writing, Govindaraju Kasivelu: reviewing, and editing References Al-Wabel MI, Al-Omran A, El-Naggar AH, Nadeem M, Usman ARA (2013). Pyrolysis temperature induced changes in characteristics and chemical composition of biochar produced from conocarpus wastes. Bioresour Technol 131: 374– 379. 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Agron Sustain Dev 36: 36. Yao CX, Joseph S, Li LQ, Pan GX, Lin Y, Munroe P, Pace B, Taherymoosavi S, Van Zwieten L, Thomas T, Nielsen S, Ye J, Donne S (2015) Developing more effective enhanced biochar fertilizers for improvement of pepper yield and quality. Pedosphere 25: 703-712. Page 10/16 Yao Y, Gao B, Chen J, Yang L (2013) Engineered biochar reclaiming phosphate from aqueous solutions: mechanisms and potential application as a slow-release fertilizer. Environ Sci Technol 47: 8700–8708. Yuan H, Lu T, Wang Y, Huang H, Chen Y (2014) Influence of pyrolysis temperature and holding time on properties of biochar derived from medicinal herb (Radix isatidis) residue and its effect on soil CO2 emission. J Anal Appl Pyrolysis 110: 277–284. Zhao L, Cao X, Zheng W, Scott JW, Sharma BK, Chen X (2016) Co-pyrolysis of biomass with phosphate fertilizers to improve biochar carbon retention, slow nutrient release, and stabilize heavy metals in soil. ACS Sustainable Chem Eng 4: 1630–1636. Zheng JF, Han JM, Liu ZW, Xia WB, Zhang XH, Li LQ, Liu XY, Bian RJ, Cheng K, Zheng JW, Pan GX (2017) Biochar compound fertilizer increases nitrogen productivity and economic benefits but decreases carbon emission of maize production. Agricult Ecosys Environ 241: 70-78. Tables Table 1 Characterization of biochar and enriched biochar-based fertilizers Physicochemical properties Physicochemical properties Proximate analysis Biochar pH EC (dSm-1) CEC (cmol/kg) BD (g/cc) MC (%) VC (%) Ash (%) FC (%) N (%) P (%) K (%) S (%) RHB 9.0 1.56 26.2 0.22 3.9 17.3 23.5 55.4 0.71 0.87 2.03 0.04 EB-1 8.07 0.15 17.0 - - - - 43.4 6.0 6.0 4.0 - EB-2 7.6 0.17 21.0 - - - - 43.7 6.0 6.0 4.0 - Table 2. Apparent nutrients recovery (%) of N, P and K under direct seeded rice. Page 11/16 Treatments Apparent Nitrogen Recovery (%) Apparent Phosphorus Recovery (%) Apparent Potassium Recovery (%) Source Fertility level Conventional fertilizers 100 % RDF 38.52 17.86 60.17 EB-1 + UEB-1 75 % RDF 41.18 15.01 48.00 100 % RDF 43.05 23.74 78.96 125 % RDF 44.49 26.76 80.05 75 % RDF 46.52 19.79 64.28 100 % RDF 49.91 29.39 83.06 125 % RDF 47.39 28.36 81.54 EB-2 + UEB-2 EB-enriched biochar; UEB-Urea enriched biochar; RDF-recommended dose of fertilizer Figures Figure 1 1a&b shows the SEM images of biochar derived from of rice husk Page 12/16 Figure 2 Cumulative (a) and Instantaneous (b) concentrations of NH4+ released Page 13/16 Figure 3 Cumulative (a) and Instantaneous (b) concentrations of NO3- released Page 14/16 Figure 4 Cumulative (a) and Instantaneous (b) concentrations of P released Page 15/16 Figure 5 Cumulative (a) and Instantaneous (b) concentrations of K+ released Page 16/16 

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Hindawi Journal of Healthcare Engineering Volume 2019, Article ID 9807619, 13 pages https://doi.org/10.1155/2019/9807619 Research Article Breast Cancer Identification via Thermography Image Segmentation with a Gradient Vector Flow and a Convolutional Neural Network Santiago Tello-Mijares ,1,2 Fomuy Woo,3 and Francisco Flores2 1 Instituto Tecnológico Superior de Lerdo, Postgraduate Department, Lerdo 35150, Mexico Instituto Tecnológico de la Laguna, Postgraduate Department, Torreón 27000, Mexico 3 Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado, Medical Familiar Unit, Torreón 27268, Mexico 2 Correspondence should be addressed to Santiago Tello-Mijares; jtello@itslerdo.edu.mx Received 2 April 2019; Revised 28 June 2019; Accepted 13 August 2019; Published 3 November 2019 Academic Editor: Vincenzo Positano Copyright © 2019 Santiago Tello-Mijares et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Breast cancer is the most common cancer among women worldwide with about half a million cases reported each year. Mammary thermography can offer early diagnosis at low cost if adequate thermographic images of the breasts are taken. The identification of breast cancer in an automated way can accelerate many tasks and applications of pathology. This can help complement diagnosis. The aim of this work is to develop a system that automatically captures thermographic images of breast and classifies them as normal and abnormal (without cancer and with cancer). This paper focuses on a segmentation method based on a combination of the curvature function k and the gradient vector flow, and for classification, we proposed a convolutional neural network (CNN) using the segmented breast. The aim of this paper is to compare CNN results with other classification techniques. Thus, every breast is characterized by its shape, colour, and texture, as well as left or right breast. These data were used for training as well as to compare the performance of CNN with three classification techniques: tree random forest (TRF), multilayer perceptron (MLP), and Bayes network (BN). CNN presents better results than TRF, MLP, and BN. 1. Introduction Breast cancer is the most common cancer worldwide among women; approximately 2 in 5 women worldwide will develop breast cancer during their lives [1]. Since 2013, breast cancer has been the leading cause of death in women [2]. The World Health Organization (WHO) estimates that by the year 2030, an estimated 27 million new cases can be expected [3]. Early detection of this disease plays an important role in reducing the mortality rate [4]; if the tumor is detected before reaching a size of 10 mm, the patient has an 85% chance of complete remission [5]. There are currently many different techniques to diagnose this pathology (mammography, ultrasound, magnetic resonance, biopsies, and more recently thermography) [6]. Mammography is currently the most common technique, but it uses ionizing radiation and is painful due to breast compression [5]. It detects cancer 8 to 10 years later than thermography [4]. In recent years, there has been a growing interest in the analysis of thermography images [7–10] to detect breast cancer. These techniques can increase productivity in the analysis of breast cancer and reduce detection errors [11]. Next, we summarize the principal computer vision works on the subject in recent years, segmentation and classification. The first task in a computer vision system is segmentation. There are various examples of segmentation work [2, 7, 12–19]. In one study, the software package ThermoMED was used to investigate the ability of thermography to detect multicentric or multifocal breast carcinomas in a preoperative setting [12]. Breast thermogram images have been segmented using a projection profile approach and by asymmetry analysis of the left and right breasts to detect 2 cancer [13]. Segmentation has also been done using by a Gaussian mixture model. The Gaussian mixture model parameters are estimated using the expectation-maximization algorithm [14]. Whole-body PET-CT and thermograms were compared in diagnosing breast cancer with breast biopsy as a standard [15]. A novel extended hidden Markov model (EHMM) was presented for optimized segmentation of breast thermograms and was compared with other segmentation techniques [7]. A blood vessel segmentation method was proposed using three enhanced images to detect possible vessel regions based on their intensity and shape [16]. A segmentation technique was proposed for thermographic images, which considers the spatial information of the pixel contained in the image [2]. Important advances in the field have been achieved in [17–19]. In one study, the tumor region was found by applying fuzzy c-means for segmentation of the hottest regions in abnormal breasts [17]. Three image segmentation methods, k-means, fuzzy c-means, and level set, were compared in [18], the level set being a more accurate approach. In [19], a novel lazy snapping method was presented for detecting hot or cold regions in medical thermographic images to segment different diseases in the breast, foot, knees, lower back, and abdomen. Next, we review related papers on thermographic breast image classification [1, 3, 4, 6, 20–30], which is the topic of our work. Two different kinds of neural network classifiers have been compared: a feedforward neural network and a radial basis function classifier [20]. Breast cancer analysis was performed using a series of statistical features extracted from the thermograms coupled with a fuzzy rule-based classification system for diagnosis [21]. Fractal analysis of breast thermal images was done to develop an algorithm [22]. The effectiveness of bispectral invariant features in the diagnostic classification of breast thermal images was evaluated, and a phase-only variant of these features was proposed. Classification was done using AdaBoost [4]. In another study [6], the diagnostic power of thermography in breast cancer was evaluated using 16 qualitative and explanatory variables and hill climbing classifiers. Araújo et al. proposed a three-stage feature extraction approach using Fisher’s criterion and minimum distance classifiers (Euclidean distance) [3]. Rotational thermography techniques were evaluated, and texture features were extracted in the spatial domain and fed to a support vector machine (SVM) for automatic classification [23]. A system was presented based on 20 gray level co-occurrence matrices with feature extraction and classification by the k-nearest neighbors method [24]. An expert system was developed based on the measured temperature gradients (ΔT) in thermograms and classified them as normal, abnormal (ΔT > 2.5, <3), and potentially having breast cancer (ΔT ≥ 3) [25]. A computer-aided detection (CAD) system was proposed with a segmentation approach based on both neutrosophic sets and the optimized fast fuzzy c-mean method [26]. Statistical, texture, and energy features were extracted and then classified by the SVM. Another method extracted Journal of Healthcare Engineering statistical features and fed them to a nearest-neighbors classifier [27]. Hot spots and warm spots have also been detected in each view and region of interest, and features were extracted from them to feed SVMs and random forests [28]. A breast cancer detection algorithm was proposed based on texture feature extraction, a Markov random field, and a modified local binary pattern. Classification was done by a decision-level fusion algorithm by means of a hidden Markov model [29]. An asymmetry approach was proposed using the detection of any type of abnormalities (MC, masses, etc.) and bilateral subtraction [30]. Another method extracts 20 characteristics of the relationship of temperatures and classifies them by sequential minimal optimization [1]. Basically, our research is one of the next logical steps in progressing thermography in the breast cancer classification field by using convolutional neural networks and also presents a novel method that has not been used for the segmentation of breasts. CNNs have recently been used in several applications, including hand-written digit recognition, face detection, face recognition, and different medical applications [31–35]. Here, we focus on breast thermography images to identify cancer by CNN. We classify these data into normal (Figures 1(a) and 1(b)) and abnormal (Figures 1(c) and 1(d)); these images were evaluated and classified by two medical experts. In this paper, we present an effective and efficient method to segment thermographic breast images and identify breast cancer for classification as normal or abnormal (without cancer or with cancer (Figure 2). The main contributions are the novel use of the combination curvature function k (cvt k) and gradient vector flow method (GVF) for breast segmentation, and for the analysis and classification of the segmented thermographic images, we proposed the use of a convolutional neural network (CNN); we also present the comparison of CNN classification results with tree random forest (TRF), multilayer perceptron (MLP), and Bayes network (BN). The paper is organized as follows. Section 2 describes the proposed segmentation and classification algorithms for breast thermographic images (Figure 2). Section 3 presents the identification results obtained by applying different classification strategies in the most difficult validation scheme (2-fold cross validation). Section 4 discusses the results and concludes the paper. 2. Materials and Methods Figure 2 illustrates the stages of the proposed segmentation algorithm and classification techniques implemented. These are detailed in the following sections. The proposed system is accomplished in four stages: image preprocessing RGB and gray input, image denoising, and curvature function k (cvt k) for initial elliptical points for the GVF and classification; we then nest the breast image segmentation by gradient vector flow snake (GVF): following first by feature extraction (shape, colour, texture, and left and right breast relation) for feeding the three classification techniques TRF, MLP, and BN in comparison with the CNN; finally, we classify the segmented images as normal or abnormal with CNN using Journal of Healthcare Engineering 3 (a) (b) (c) (d) Figure 1: Input images. (a, b) Thermographic breast image without cancer. (c, d) Thermographic breast image with cancer. Preprocessing Breast segmentation by GVFS Classification Breast feature extraction Input image Segmented and classified breast Figure 2: Proposed GFV segmentation method for breast cancer identification. only the segmented regions of interest obtained by cvt k and GVF. The proposed technique is applicable to all breasts modifying the parameters of the cvt k and GVF. 2.1. Preprocessing for Initial Elliptical Points. The input RGB breast images are for feature extraction, and the input gray breast images are for preprocessing and segmentation (Figure 2, image provided by Silva et al. [11]). The input gray breast image is first denoised with a Gaussian filter (3 × 3). The perceptual linearity makes it more suitable for gradient vector flow snake implementation for the breast region segmentation, which is the following step. We start from the observation that the breasts are anatomically elliptical. Boundary analysis used the initial snake for the GVF. Boundary analysis using the curvature function k identified the salient points on the curve. The input gray thermographic breast image filtered was first converted to binary using a predefined threshold of 0.25. Later, these were defined as left and right margins via canny edge detection (lines in Figure 3(a)). These two margins or lines represent two closed object boundaries via a sequence of points C � {(xn, yn)}, where xn � x(tn) and yn � y(tn). The curvature at a point on this planar curve (defined by the sequence of boundary points) is the rate of change of the angle with respect to arc length k � dθ/ds. Here, s is the arc length parameter. The curvature is a local geometric property of the curve. The tangent vector T shown in Figure 3(a) is defined by T � 􏼂 x_ y_ 􏼃T . The normal vector N, which is perpendicular T to the tangent vector, is given by N � 􏼂 − y_ x_ 􏼃 . The tangent of the angle θ at (x(tn), y(tn)) is given by _ y. _ It can then be shown that the curvatan θ � dy/dx � x/ ture k of the parametric curve can be written as € x_ 2 + y_ 2 )3/2 ). cvt k � x€y_ − (x_ y/( Figure 3(b) shows the boundaries on which the positive and negative curvatures are labelled with different red markers in the right and left breasts. The two graphs shown in Figure 3(d) depict the curvature function cvt k for the right and left breasts. In the cvt k of the right breast, we emphasize the positive peaks. The negative peaks are emphasized in the cvt k of the left breast; peaks of interest are in red in both figures. Note that in the right breast, the cvt k is positive when the boundary line is concave. It is negative in 4 Journal of Healthcare Engineering t1 T (X(tn), Y(tn)) θ N t6 (a) (b) (c) Curvature function of right breast 0.02 cvt k 0 –0.02 –0.04 –0.06 20 30 40 50 tn 60 70 80 70 80 Curvature function of left breast 0.06 cvt k 0.04 0.02 0 –0.02 20 30 40 50 tn 60 (d) Figure 3: Initial elliptical points for GVF. (a) Left and right margins or lines. (b) Boundaries from curvature function. (c) Initial elliptical points obtained from cvt k for GVF. (d) Curvature function of right breast and left breast (Video 1 (MPEG, 169 KB) of supplementary materials). the left breast when the boundary curve is convex. These features indicate the initial elliptical points of interest for the gradient vector flow snake. Thus, the curvatures of the boundary curves carry unique signatures that we utilized for right and left breast identification via the morphological contraction of these two elliptical objects defined in red (Figure 3(c)). 2.2. Breast Segmentation by Gradient Vector Flow Snakes. Once an image is preprocessed, the list of initial points is then initialized for left and right breasts (Figure 4); we start from the observation that regions belonging to a breast should be elliptical and nearly similar. Thus, the list of initial points forms ellipses. These two ellipses can work like initial points for the GVF snakes may be applied. We followed the GVF method of our previous work, concerning automated pollen grain detection and classification from earlier microscopic prepared images [36]. Traditional snakes are curves (v(s) � [x(s), y(s)], s ∈ [0, 1]) defined within the domain of an image; it can move itself under the influence of internal forces coming from within the curve itself and external forces computed from the image data as first introduced by Kass et al. [37]. The GVF improves the capture range of the contours obtained by the binary image. Xu and Prince [38] proposed an improved snake to obtain better performance for image segmentation (Figure 4). The formulation of a GVF is valid for gray images as well as binary images; however, we used gray images as seen in Figures 2 and 3. To compute GVFS, an edge-map function is first calculated using a Gaussian function. The initial values are based on a priori knowledge and several experiments for both breasts. α specifies the elasticity of the snake, and this controls the tension in the contour by combining with the first derivative term (alpha � 0.20). β specifies the rigidity in the contour by combining with the second derivative term (beta � 0.20). c specifies the step size (gamma � 1.00). κ acts as the scaling factor for the energy term (kappa � 0.1). The wEline weighting factor is used for the intensity-based potential term (wl � 0.01). The wEedge weighting factor is for the edge-based potential term (we � 0.40). The wEterm weighing factor is for the termination potential term (wt � 0.01). The user then specifies the number of iterations for which the contour’s position is to be computed with iterations of 5000. An edge-map function and an approximation of its Journal of Healthcare Engineering 5 (a) (b) (c) (d) Figure 4: GVF segmentation of the breast region of interest. (a) Initial, (b) 200 iterations, (c) 5000 iterations, and (d) segmented CNN input image (Video 2 (MPEG, 489 KB) of supplementary materials). gradient are then given. The GVFS is computed to guide the deformation of the snake at the boundary edges. Figure 4 shows the results of variations in breast segmentation and classification based on RGB thermographic breast images. The segmented CNN input image is shown in Figure 4(d). This phase is designed to maximize recall and avoid false negatives of TRF, MLP, and BN. This is a critical factor in medical imaging. The objective of the following CNN phases (Section 2.4) is to maximize the precision and remove, or at least to identify, false candidates. The specific values of the a priori restrictions are quite flexible because they should avoid missing true positives. We first obtained the initial results, which are presented in the following section (Section 2.3), and compared the results of TRF, MLP, and BN with those of CNN. 2.3. Feature Extraction and Classification. This part aims to characterize the segmented breast with a feature vector that helps identify cancer in the thermographic breast images. These breast regions are then mapped on the R ∗ G ∗ B ∗ colour model image for feature extraction. The selected features can be grouped into four categories (Table 1). 2.3.1. Shape Descriptors. The shape differences between left and right breast give clues for classification. First, the area (A) of the grain is determined by counting the number of pixels within the border, and the perimeter (P) is the length Table 1: Summary of descriptors. Shape Area Perimeter Roundness Compactness First-order texture Average Median Variance Standard deviation Entropy Second-order texture Contrast descriptor Correlation Energy Local homogeneity Relation context Euclidian distance Bhattacharyya distance Difference P� A � nPixels 􏽱��������������������� (xi − xi− 1 )2 + (yi − yi− 1 )2 R � 4π(A/P2 ) C � A/P2 μ � 1/ij􏽐i,j p(i, j) m � L + I((N/2) − F/f) σ2 �􏽱 1/ij􏽐 i,j ((p(i, j) − μ)) ���������������� σ � 1/ij􏽐i,j (p(i, j) − μ) S � − 􏽐i,j p(i, j)log p(i, j) CM � 􏽐i,j |i − j|2 c(i, j) r � 􏽐i,j (i − μci )(j − μcj )c(i, j)/σ ci σ cj e � 􏽐i,j c(i, j)2 HL � 􏽐i,j c(i, j)/(1 + |i − j|) 􏽱��������� 2 ED � 􏽰(V r − Vl ) �������� BD � (Vr × Vl ) D � abs(Vr − Vl ) of the border. The regions A and P can be used as descriptors because of the differences in size between breasts; this is a medical parameter of interest. The roundness (R) is defined as the multiplication of 4π and A over P2. If R � 1, then the object is circular. The compactness (C) is defined as the result of A over P. Each breast (left and right) gave eight terms: Al, Ar, Pl, Pr, Rl, Rr, Cl, and Cr. 6 2.3.2. First-Order Texture Descriptors. One way to discriminate between different textures is to compare R∗ , G∗ , and B∗ levels using first-order statistics. Red indicates high temperatures related to breast cancer. First-order statistics are calculated based on the probability of observing a particular pixel value at a randomly chosen location in the image. They depend only on individual pixel values and not on the interaction of neighboring pixel values. The average (µ) is the mean of the sum of all intensity values in the image. The median (m) represents the value of the central variable position in the dataset of sorted pixels. The variance (σ 2) is a dispersion measure defined as the squared deviation of the variable with respect to its mean. Standard deviation (σ) is a measure of centralization or dispersion variable. Entropy (S) of the object in the image is a measure of content information. For both left and right breasts (µl, µr, ml, mr, σ 2l , σ 2r , σ l, σ r, Sl, and Sr), R∗, G∗, and B∗ levels gave 30 features. 2.3.3. Second-Order Texture Descriptors. Haralick’s graylevel co-occurrence matrices [39] have been used very successfully for biomedical image classification [40, 41]. Out of 14 features outlined, we first considered four texture features suitable for our experiment. We propose to use the co-occurrence matrix for the entire R∗G∗B∗ colour model. Contrast descriptor (CM) is a measure of local variation in the image. It is a high value when the region within the range of the window has high contrast. Correlation (r) of the texture measures the relationship between the different intensities of colours. Mathematically, the correlation increases when the variance is low, suggesting that the matrix elements are not far from the main diagonal. Energy (e) is the sum of the squared elements in the matrix of co-occurrence of gray level, also known as the uniformity or the second element of the angular momentum. Local homogeneity (HL) provides information on local regularity of the texture. The value of the local homogeneity is higher when the elements of the co-occurrence matrix are closer to the main diagonal. Both the left and right breasts (CMl, CMr, rl, rr, el, er, HLl, and HLr) were classified via the R∗ , G∗ , and B∗ levels to give 24 features. 2.3.4. Relation Context Features. Relation breast context features are selected to capture the value of the relation between left and right breasts in the R∗ , G∗ , and B∗ levels considering the asymmetries between the breasts as an abnormality indicator. This yields values for relationships with respect to the left and right features: Euclidean distances, Bhattacharyya distance (BD), and absolute differences (D). The shape gave 12 features, the first-order texture gave 45 features, and the second-order texture gave 36 features. Thus, there were 93 relation context features. 2.3.5. Classification. The main aim of this work is to compare the proposed segmented image and the CNN classification with the characterization and classification of breasts using three classification techniques. In order to classify the segmented breast into normal or abnormal, Journal of Healthcare Engineering identify cancer, and obtain final classification results, we explored the use of three different classification approaches implemented in Weka (Waikato Environment for Knowledge Analysis) [42, 43]: tree random forest (TRF) [44], multilayer perceptron (MLP), and Bayes network (BN). The experimental results obtained for these three classification techniques are compared with CNN classification (see results in Figure 5, Table 2, and Section 3). 2.4. CNN Classification. We here present an effective and efficient CNN classification system (Figure 6). Originally proposed by LeCun et al. [45], a CNN is a neural network model with three key architectural ideas: local receptive fields, weight sharing, and subsampling in the spatial domain. A CNN consists of three main types of layers designed to obtain the feature maps: spatial convolution layers (Cl), subsampling pooling layers (Sl), and fully connected layers (Fl)—l is a layer index. We use a CNN based on previous studies designed to process two-dimensional (2D) images [46]. Cl and Sl layers are 2D layers, whereas the Fl layer and output are 1D layers. The motivation is that a CNN is advantageous for only breast thermography image in that it is hierarchical (with multiple layers for more compactness and efficiency) and invarianceredundant (for position, size, luminance, rotation, poseangle, noise, and distortion). We propose an efficient method to classify the segmented breast thermography image with a gradient vector flow to feed the CNN. We demonstrate that a classification method using the segmented breast to feed CNN is more robust and efficient than conventional state-of-the-art (SoA) methods using only classical features and classification techniques (Section 2.3.5). Our main contributions are as follows: the generation of segmented input images, capturing relevant breast information, and training and feeding the CNN for the comparison and evaluation of several classification strategies to confront the classification problem—TRF, MLP, and BN. For every thermography breast image, we generate a segmented image input to capture the semantics of the breast. These image are modified to 277 × 277 × 3 RGB images to feed the CNN (Figure 6). 3. Experimental Results 3.1. Dataset Description. The dataset includes 63 thermographic images (35 normal and 28 abnormal) in RGB colour format and in JPEG image format with a size of 680 × 480 × 3 as kindly provided by Silva et al. [11] and can be downloaded from [47]. There are 155 × 35 normal features and 155 × 28 abnormal features to test and train TRF, MLP, and BN obtained from 63 segmented CNN input images (35 normal and 28 abnormal). However, the proposed method can be easily adapted to different thermographic breast images. Figure 1 shows example images of the dataset, and Figures 7 and 8 show the ground truth from two medical experts, segmentation and classification results of these images. The ground-truth data for the segmentation and classification of breast tissue and their degree of alteration with respect to Journal of Healthcare Engineering 7 Normal TRF 100.0% 35 0.0% 0 0.0% 0 100.0% 28 Abnormal Normal Output class Output class CNN Normal 88.2% 30 17.2% 5 Abnormal 11.8% 4 82.8% 24 Abnormal Target class Normal (a) Abnormal Target class (b) Normal BN 100.0% 28 20.0% 7 0.0% 0 80.0% 28 Abnormal Normal Output class Output class MLP Normal 82.9% 29 21.4% 6 Abnormal 17.1% 6 78.6% 22 Abnormal Target class Normal (c) Abnormal Target class (d) Figure 5: Confusion matrices of (a) CNN, (b) TRF, (c) MLP, and (d) BN. Table 2: Quantitative classification results (%). Technique TPR 100 85.71 80 82.85 CNN TRF MLP NV PPV 100 85.71 80 82.85 External quality indicators F1 or HM SPC NPV 100 100 100 86.95 85.71 85.71 88.88 100 100 82.85 78.57 78.57 FDR 0 14.28 20 17.14 FPR 0 17.85 25 21.42 ACC 100 85.71 88.88 80.95 AUC 100 85.71 100 78.57 ACC: accuracy; AUC: area under the receiver operating characteristic curve; F1: F1 score; FDR: false discovery rate; FPR: fall-out or false-positive rate; HM: harmonic mean; NPV: negative predictive value; PPV: precision or positive predictive value; SPC: specificity or true-negative rate; TPR: sensitivity, recall, or true-positive rate. (55 – 3)/2 + 1 = 27 4 CONV 3×3 pad = 1 384 kernels 38 4 27 55 GVF segmented image for CNN input 38 27 (13 + 2 ∗ 1 – 3)/1 + 1 = 13 13 13 CONV 3×3 pad = 1 256 kernels (13 + 2 ∗ 1 – 3)/1 + 1 = 13 13 13 6 25 6 25 (27 + 2 ∗ 2 – 5)/1 + 1 = 27 27 CONV 3×3 pad = 1 384 kernels (13 + 2 ∗ 1 – 3)/1 + 1 = 13 (27 – 3)/2 + 1 = 13 13 27 13 13 Overlapping Max POOL 3×3 stride = 2 13 Overlapping Max POOL 3×3 stride = 2 6 (227 – 11)/ 55 4 + 1 = 55 CONV 5×5 pad = 2 256 kernels 25 11 6 11 25 227 Overlapping Max POOL 3×3 stride = 2 96 CONV 11 × 11 stride = 4 96 kernels 96 227 (13 – 3)/2 + 1 = 6 6 FC FC 9216 Figure 6: Convolutional neural network. 4096 4096 Classification by CNN as abnormal 8 different temperature levels were obtained from two oncologists via breast localization. We then present very similar segmentation results to those of medical experts according to the Zijdenbos Similarity Index with excellent result classification. 3.2. Quality Indicators of Thermographic Breast Cancer Image Classification. Several quality indicators have been obtained to quantitatively assess the breast classification results and the performance of the CNN, TRF, MLP, and BN techniques. We have divided them into final and external quality indicators, which evaluate the final segmentation results and are useful for external comparison with other works, and internal quality indicators, which are useful for evaluating the internal behavior of the proposed classification options. For external indicators, let P be the number of normal breasts in the dataset, and let TP, FP, and FN be the number of true positives, false positives, and false negatives, respectively (Figure 5). We then define the following (see Table 2): sensitivity, recall, or true-positive rate: TPR � TP/P; precision or positive predictive value: PPV � TP/TP + FP; false discovery rate: FDR � FP/FP + TP; and the F1 score, overlap, or harmonic mean of TPR and PPV: F1 � 2 ∗ TP/ 2 ∗ TP + FP + FN, HM � 2 ∗ TPR ∗ PPV/TPR + PPV. As the proposed algorithm will classify breast temperature regions of interest, which are then characterized and separated into normal and abnormal, we can further evaluate the classification performance of the four selected classification schemes via internal indicators. Let N be the number of abnormal breasts with cancer resulting from the application of the proposed method to the complete dataset, and let TN be the number of true negatives after classification (Figure 5). We can then define the following (see Table 2): specificity or true negative rate: SPC � TN/N; negative predictive value: NPV � TN/TN + FN; accuracy: ACC � TP + TN/TP + FP + TN + FN; fall-out or false-positive rate: FPR � FP/N; and the area under the receiver operating characteristic curve: AUC. 3.3. Quantitative and Qualitative Evaluation of Thermographic Breast Cancer Image Classification. There were 63 thermographic images (35 normal and 28 abnormal) for a total of 155 × 35 normal features and 155 × 28 abnormal features. The segmented breasts and the feature extraction phases for the described dataset include a collection of 155 total extracted features from 63 different breast images (35 normal breast and 28 expected abnormal breast). A 155dimension feature vector extracted from shape, first-order texture, second-order texture, and relation context features characterized each segmented breast. As mentioned, three representative classification techniques were explored (BN, MLP, and TRF) using the toughest but most realistic classification experiment involving 2-fold cross validation schemes (s � 2) for training and testing. Figure 5 and Table 2 summarize the quantitative results of CNN, BN, MLP, and TRF. CNN achieved the best results in 2-fold cross validation where the dataset is divided into two equal parts: the first part is used for training and the second is used for testing. Journal of Healthcare Engineering This was later switched: the second part was used for training and the first part for testing. This proves that these classifiers are reasonable for the classification of breast thermographic images and confirms the advantages of CNN over other state-of-the-art classifiers. Qualitative results of breast classification are shown in Figures 7 and 8 with white (for ground truth and segmentation) and red and blue regions superimposed over correctly detected normal breast (TP, in blue in Figures 7(m)–7(p)) and correctly classified abnormal breast (TN, in red in Figures 8(m)–8(p)). These illustrate the good performance of the feature extraction and classification phases. The results show that the proposed classification method can successfully classify breast even in challenging environments. 3.4. Quantitative and Qualitative Evaluation of Nuclei Segmentation. Two medical experts defined a region around both breasts to define ground truth (histologically confirmed diagnosis) comparison (see Figures 7(a)–7(h)) and 8(a)–8(h)). Thus, good segmentation and precise breast cancer classification are both desired. To assess segmentation quality, we compared the region associated with a correctly identified breast and the corresponding region in the ground truth. The comparisons were quantified using the Zijdenbos Similarity Index, ZSI � 2 ∗ |A1 ∩ A2|/(|A1| + |A2|), where A1 and A2 refer to the compared regions and are both binary masks. A ZSI value greater than 0.75 indicates excellent agreement [48]. The ground truth includes segmentation data from two different experts referred to as GT1 and GT2. The two expert results comprise regions associated with correctly identified breast segmentation by GVF. Table 3 summarizes the statistics for the ZSI obtained for every possible pair of experts (A1 and A2 comparison). The ZSI for the GVF compared with the GT1 had a mean of 0.8177 and a standard deviation of 0.0173; with GT2, the mean was 0.8229 and the standard deviation was 0.0094. This proves that the proposed segmentation approach gives similar results as those obtained manually by an expert. 3.5. Comparative Discussion. Publicly accessible datasets or evaluation scenarios that allow for a fair comparison among methods are lacking, and the code for reported methods is unavailable. Thus, we have chosen to present just our results on thermographic breast image classification. One study [20] showed the feasibility of applying an ANN for the early detection and differentiation of abnormal patient states in health screening; their classifying systems are effective to the tune of more than 92% accuracy. Another study [21] concluded that the presented approach is indeed useful as an aid for the diagnosis of breast cancer and should prove even more powerful when coupled with another modality such as mammography; their approach provides a classification accuracy of about 80%. Yet another study [22] suggested that fractal analysis may potentially improve the reliability of thermography in breast tumor detection, with an accuracy of 90%. It has been shown that higher-order Journal of Healthcare Engineering 9 (a) (b) (c) (d) (e) (f ) (g) (h) (i) (j) (k) (l) (m) (n) (o) (p) Figure 7: Thermographic breast image without cancer (Video 2 (MPEG, 489 KB) of supplementary materials). Ground truth from the first medical expert (a–d), ground truth from the second medical expert (e–h), segmentation (i–l), and classification results (m–p). spectral features are capable of differentiating between different classes such as malignant, benign, and normal tissue in breast thermograms [4]; malignant cases are detected with 95% accuracy. The framework of Bayesian networks provides a good model for analyzing thermographic breast images [6], obtaining an accuracy of 71.88%. A new feature extraction approach was presented for breast thermography classification [3]. This approach combines morphological, mathematical, and symbolic data analysis operators to discriminate different classes such as malignant, benign, and cyst tissue in breast thermograms; they reported a 85.7% of sensitivity and 86.5% of specificity to the malignant class. A pilot study [23] evaluated the potential of rotational thermography for automatic detection of breast abnormalities from the perspective of cold challenge; the accuracy of the classification system is found to be better than 83%. The goal of another work [24] was to compare the classification results of three different classifiers (SVM, k-NN, and Naive Bayes) and use GLCM features extracted from each thermography image; they obtained the accuracy ratio of 92.5%, which corresponded to true positive fraction of 78.6% at a false positive fraction of 0%. In another pilot study [25], digital infrared thermal imaging showed promising results and is thus well suited as a screening tool, obtaining a sensitivity of 97.6%, specificity of 99.17%, positive predictive value of 83.67%, and negative predictive value of 99.89%. Its use in combination with other laboratory and outcome assessment tools could lead to a significant improvement in the management of breast cancer. Other results [12] indicate that thermography has the necessary sensitivity to effectively and inexpensively provide such an assessment; a sensitivity of 100% was claimed in their work (full train and full test). Another study [26] used several features (statistical, texture, and energy) with the SVM to detect normal and abnormal breast tissue; their system was achieving an excellent result of 100% using leaveone-out cross validation. Other results [27] showed that 10 Journal of Healthcare Engineering (a) (b) (c) (d) (e) (f ) (g) (h) (i) (j) (k) (l) (m) (n) (o) (p) Figure 8: Thermographic breast image with cancer (Video 2, MPEG, 489 KB). Ground truth from the first medical expert (a–d), ground truth from the second medical expert (e–h), segmentation (i–l), and classification results (m–p). Table 3: ZSI statistics for segmentation results and ground truth. A1 GVF A2 GT1 0.8177 ± 0.0173 GT2 0.8229 ± 0.0094 GT: ground truth; GVF: gradient vector flow segmentation result; ZSI: Zijdenbos Similarity Index. using simple texture descriptors in combination with a nearest-neighbors classifier can detect the early onset of breast tumors in women of any age, where abnormal breasts were identified with an accuracy of 94.44%. A potential breakthrough was indicated in thermographic screening for breast cancer [28]; they were able to achieve around 99% specificity while having 100% sensitivity. Other results [29] indicated that useful features of texture can be extracted with MRF models, LBPc, and LBPe and decision-level fusionbased classification using HMM on thermography images to achieve 87% accuracy. A CADx methodology dedicated to the creation of patient features combined the information of contralateral asymmetry and different views into single feature [30]; an area under the roc curve of 73.8% and 76.7% was achieved. The WEKA software SMO classifier obtained more expressive results regarding the diagnosis of breast abnormalities [1], achieving 93.42% accuracy, 94.73% sensitivity, and 92.10% specificity for the cancer class in a binary (cancer versus noncancer) analysis. In light of these studies, we can confirm to some extent that our approach is valid based on our CNN classification results (TPR � 100% and PPV � 100% for a dataset containing 73 breast images, using 2-fold cross validation) in comparison with other similar “state-of-the-art” studies. 4. Conclusion The main objective of this work is to make scientific contributions to a biomedical system for the acquisition of thermographic images of breasts via image processing. This Journal of Healthcare Engineering provides a prediagnosis of breast cancer via GVF in combination with CNN. This paper proposes a novel method of initial selection of areas of interest in the chest through the analysis of the cvt k in both the right and left chest. The initial regions of interest of both breasts then feed the GVF technique to extract the characteristics for an accurate classification of the segmented regions. Finally, this determines the difference between the normal cases (without cancer) and abnormal cases (with cancer). This work shows that a classification method that uses the combination of breast segmentation by GVF and applying CNN classification can be robust and efficient. Our main contributions include the novel segmentation via GVF of the region of interest of the thermographic image of the sinuses; segmentation of these input images to capture relevant information from the breasts to train and feed CNN, BN, MLP, and TRF with the segmented image or with feature extraction; the generation of a set of representative data with ground-truth data by specialist physicians to compare with our segmentation technique; and the evaluation of four classification strategies (CNN, BN, MLP, and TRF). We compared our data to the state of the art and observed that this approach gave results between 80% and 100% for TPR, SPC, and ACC. Thus, this approach improves outcomes and accuracy. We demonstrated that a combination of GVF and CNN can detect breast cancer via the classification of thermographic images. The best results were obtained using CNN classifiers (100% TPR, SPC, and ACC). These results validate the novelty and quality of the proposed method. Future work will include a secondary framework to objectively compare our results. Data Availability The data used to support the findings of this study are included within the supplementary material. Conflicts of Interest The authors declare that there are no conflicts of interest regarding the publication of this paper. Funding This study was supported by internal grant “ITSLerdo #002POSG-2018.” Supplementary Materials Figures 1–7: original images of Figures 1 to 7 in png format. MATLAB breast data: first run “AlexCNNbreast.m” code. Segmentation results of the breast images extracted from the gradient flow method (63 images in bmp format on folder “myImagesSEGMENTEDbreast” divided into normal and abnormal) to feed the convolutional neural network in Matlab2018a (“AlexCNNbreast.m” code to obtain the classification results and CNN models); the two CNN models from the 2-fold cross validation (“myNet_s1.mat” and “myNet_s2.mat”) that obtained 100% of TPR, SPC, and ACC; 11 plotConfMat.m code to obtain the confusion matrices of CNN, TRF, MLP, and BN. Video results of Figures 3 and 4: VIDEO 1 of Figure 4 in mp4 format—this video describes the initial elliptical points for gradient vector flow using the curvature function k of right and left breasts; VIDEO 2 of Figure 4 in mp4 format—this video describes the gradient vector flow segmentation of the breast region of interest. WEKA breast data features: 155 × 63 classical features in Weka for TRF, MLP, and BN results. Run “BreastDatasetFeatures.arff” for obtaining the classification results. (Supplementary Materials) References [1] J. H. Vasconcelos, W. P. dos Santos, and R. C. F. de Lima, “Analysis of methods of classification of breast thermographic images to determine their viability in the early breast cancer detection,” IEEE Latin America Transactions, vol. 16, no. 6, pp. 1631–1637, 2018. [2] M.-A. Dı́az-Cortés, N. Ortega-Sánchez, S. Hinojosa et al., “A multi-level thresholding method for breast thermograms analysis using dragonfly algorithm,” Infrared Physics & Technology, vol. 93, pp. 346–361, 2018. [3] M. C. Araújo, R. C. F. Lima, and R. M. C. R. de Souza, “Interval symbolic feature extraction for thermography breast cancer detection,” Expert Systems with Applications, vol. 41, no. 15, pp. 6728–6737, 2014. [4] M. EtehadTavakol, V. Chandran, E. Y. K. Ng, and R. 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Advances in Obesity Weight Management & Control Review Article Open Access Obesity and diabetes: interrelationship Abstract Volume 8 Issue 2 - 2018 Diabetes epidemic can be attributed to the increasing incidence of obesity, especially in India. It is estimated that about 60-90% of all the patients with type 2 diabetes are obese (BMI≥30kg/m2) or overweight (BMI≥25kg/m2). A number of mechanisms involved in the pathogenesis of obesity have been proposed which play an important role in the development of diabetes by causing insulin resistance or hypersecretion of insulin. Excessive storage of fat in obese people leads to the release of excessive fatty acids resulting in insulin resistance and hyperglycemia. Insulin resistance is also a consequence of elevated secretion of cytokines (TNF-α, IL-6, complement C3, MIF and leptin) by the adipose tissue resulting in the development of diabetes. There is a close association of diabetes and obesity with low plasma concentrations of adiponectin however, further experimental studies are required to establish the role of adiponectin. Development of both the diseases can be prevented to a large extent by increasing physical activities and maintaining a healthy weight. We undertook this review to study the interrelationship between diabetes and obesity. Samreen Siddiqui Independent researcher, India Correspondence: Samreen Siddiqui, Independent researcher, M-301 Taj Enclave, Geeta Colony, Link Road, Delhi-110031, India, Tel 9582865898, Email sid.samreen@gmail.com Received: February 28, 2018 | Published: April 26, 2018 Keywords: obesity, type 2 diabetes, adipokines, cytokines Introduction Diabetes is one of the major health problems affecting large number of individuals and has reached epidemic proportion worldwide. The World Health Organization (WHO) has projected that around 300 million people will suffer from diabetes by 2025.1 Among the well-known risk factors of diabetes one of the most important and major risk factor is obesity (BMI≥30kg/m2),2 whose prevalence is also rising at a higher rate in developing countries, including India.3,4 Around 60-90% of all patients with type 2 diabetes are obese.5,6 The increasing incidence of diabetes can thus be attributed to the global epidemic of obesity.7 A number of clinical studies have reported an association between obesity and insulin resistance in adults as well as children8–10 and reports are also present which suggest that weight loss is associated with a decrease in insulin concentration and an increase in insulin sensitivity in adults and adolescents.11,12 This comorbid condition of obesity in patients with type 2 diabetes is very common and is often termed as “Diabesity”.13,14 Sedentary lifestyle and changing food habits could be the main reasons for continuously increasing incidence of obesity and diabetes both in the urban as well as the rural areas in India. The rising prevalence of these two diseases is of concern as they may act as major risk factors for other fatal conditions like coronary artery disease (CAD). The aim of this review article was to study the relationship between obesity and diabetes. This is a general review and we reviewed fifty research papers before writing this paper. eventually leads to the release of elevated levels of fatty acids (FFAs) from enhanced lipolysis. The release of these FFAs then induces lipotoxicity, as lipids and their metabolites create oxidant stress to the endoplasmic reticulum and mito­chondria. This affects adipose as well as non-adipose tis­sues resulting in insulin-receptor dysfunction. The consequence is an insulin-resistant state which then cre­ ates hyperglycemia with compensated hepatic gluconeo­genesis.17–19 Insulin resistance is a key factor for type 2 diabetes. FFAs also decrease utilization of insulin-stimulated muscle glucose, contributing further to hyperglycemia. Lipotoxicity from excessive FFAs also decreases secretion of pancreatic β-cell insulin, which eventually results in β-cell exhaustion (Figure 1). Moreover, excess adipocytes release inflammatory adipokines (TNF-α, IL-6, complement C3, leptin and MIF), which, along with free fatty acids, provide the pathophysiologic basis for comorbid conditions associated with obesity such as insulin resistance and type 2 diabetes. Along with fatty-acid lipotoxicity, visceral adipokines also contribute to the adipokine inflammatory injury that leads to pancreatic β-cell dysfunction, which, in turn, decreases insulin synthesis and secretion.20,21 Adipocytes also stimulate fat-associated macrophages that also secrete monocyte chemoattractant protein 1 (MCP-1), macrophage migration inhibiting factor (MMIF), and resistin, all of which decrease insulin sensitivity (Figure 2) (i.e. enhance insulin resistance).22,23 Pathophysiology of obesity and type 2 diabetes Pathogenesis of obesity appears to play a central role in the dysregulation of cellular mechanism that accounts for insulin resistance, which is the state of reduced responsiveness of liver, muscle and adipose tissue to insulin in type 2 diabetes. The stored fat is required for survival during nutritionally deprived states, however during state of prolonged abundance of food, excessive fat storage results in obesity.15,16 This excessive storage of fat that creates obesity Submit Manuscript | http://medcraveonline.com Figure 1 Pathway showing development of diabetes due to obesity. Adv Obes Weight Manag Control. 2018;8(2):155‒158. 155 © 2018 Siddiqui. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially. Obesity and diabetes: interrelationship Copyright: ©2018 Siddiqui 156 (visceral) adiposity. Excessive deposition of lipid in muscle and liver also enhances the risk of type 2 diabetes through mechanisms of intracellular lipotoxicity. Genetic factors linking obesity and diabetes Figure 2 Adipokines regulating insulin sensitivity Obesity and diabetes interlinked A strong relationship between obesity and the onset of diabetes has been reported in a number of studies. Research has shown that people carrying more weight particularly around the tummy are more insulin24–27 resistant and may struggle to achieve good diabetes control.10,28,29 A number of mechanisms have been proposed to link obesity and insulin resistance which predispose to diabetes and includes increased production of adipokines or cytokines including tumor necrosis factor-α, resistin and retinol-binding protein 4.30 Excess body fat and particularly visceral fat release increased amounts of FFAs in the blood. Elevation of FFAs levels directly affects insulin signaling and causes the liver and skeletal muscles to shift towards greater oxidation of FFAs for energy production and a relative inhibition of enzymes in the glycolytic cascades. As a result the capacity of liver and skeletal muscles cells to absorb and metabolize glucose decreases. Also, the tissues capacity to store glucose as glycogen decreases and the cells accumulate more triglycerides instead of glycogen. Further, in an Indian the body fat percentage is significantly higher than a western counterpart with similar BMI and blood glucose level. It has been hypothesized that excess body fat and low muscle mass may explain the high prevalence of hyperinsulinemia and the high risk of type-2 diabetes in Asian Indians.31 The risk of diabetes increases exponentially as BMI increases above about 25 kg/m2.32,33 In a large cross-sectional study in middle aged Indians, a BMI>23 was found to be associated with increased risk for type 2 diabetes.34 Visceral fat increases the risk of diabetes by favouring insulin resistance. Patients with diabetes are usually advised to increase their physical activity and reduce weight. Prolonged duration of obesity also have deleterious effects on glucose homeostasis like increased resistance to glucose disposal and decreased secretion of insulin. Resistance to glucose disposal is strongly associated with obesity and results in high fasting and postload serum insulin concentrations. Prolonged duration of obesity could conceivably worsen this resistance.35 Although excess fat in any region of the body is associated with increased risk of type 2 diabetes, it is generally held that an accumulation of abdominal fat (‘central’ obesity), as indicated by an increased waist: hip ratio is an independent risk for type 2 diabetes irrespective of the extent of obesity.36,37 This is mainly attributed to increased intra-abdominal Obesity as well as diabetes, both are the examples of multifactorial diseases that arise through the interaction of multiple genetic and environmental factors. There has been evidences present which establish the genetic link between obesity and diabetes. Genomewide association scans (GWAS) and candidate gene approaches have identified 40 genes associated with type 2 diabetes and a similar number, although largely different, with obesity. Most type 2 diabetes genes appear to be related to b-cell dysfunction, with many fewer involved in pathways related to insulin resistance independent of obesity.38–41 A growing understanding of genetics and cellular function of the b-cell can identify potential mediators predisposing obese individuals to type 2 diabetes and further may provide insights for the development of new therapeutic agents. Although numerous diabetes and obesity associated genes have been identified, the known genes are estimated to predict only 15% of type 2 diabetes and 5% of obesity risk.42,43 Recent genome-wide studies have shown multiple loci on chromosomes which affect the obesity-related phenotypes.44 It can be speculated that the susceptibility to type 2 diabetes and obesity might also partly be due to shared genes. By comparing all of the published genome scans for type 2 diabetes and obesity, five overlapping chromosomal regions for both diseases have been identified and by analysing these five susceptibility loci for type 2 diabetes and obesity, 27 functional candidate genes have been pinpointed that are involved in eating behaviour, metabolism and inflammation. These genes might reveal a molecular link between the two disorders.45 By comparing the defined obesity-relevant pathways and Non-insulin dependent diabetes mellitus (NIDDM)- relevant pathways, it has been found that obesity-relevant pathways contains a gene set related to the insulin receptor, and coincidentally, there is a NIDDM-relevant gene set containing genes 2-fold up-regulated by insulin. Other than that, all relevant pathways in obesity and NIDDM are literally different.46 Co-relation of adiponectin, obesity and diabetes Adiponectin is a novel fat protein secreted by adipose tissue and abundantly present in the circulation in humans. It has been hypothesized that this protein has a role in the pathogenesis of obesity and type 2 diabetes and that lower plasma levels of adiponectin are predictive of type 2 diabetes and found in patients with diabetes as well as in obese people.29,47–49 A review done by Hussain et al.,50 has reported that concentrations of adiponectin may be down regulated by weight gain. Diabetes and obesity both are associated with low plasma adiponectin concentrations and hypoadiponectinemia in obese people is in large part attributable to insulin resistance.51 A previous study in Japanese individuals have shown that the plasma adiponectin concentration is negatively correlated with body mass index (BMI) and therefore found to be lower in obese than in lean subjects,52 which is also the case in Indian population.53 However, the mechanism behind this close association between plasma concentration of adiponectin and insulin sensitivity is still unknown. Further experimental studies are required to study the molecular link between plasma levels of adiponectin and the pathogenesis of obesity and diabetes. Citation: Siddiqui S. Obesity and diabetes: interrelationship. Adv Obes Weight Manag Control. 2018;8(2):155‒158. DOI: 10.15406/aowmc.2018.08.00233 Copyright: ©2018 Siddiqui Obesity and diabetes: interrelationship Conclusion Obesity and diabetes which have become major health problems in India as well as globally are closely linked together, obesity serves as a major risk factor for type 2 diabetes and weight gain management can reduce the risk of diabetes to a larger extent. Urbanization, changing life style & food habits are the main reasons for increasing obesity in India and consequently responsible for increasing incidence if diabetes. Increasing physical exercise and maintaining ideal weight can lower the chances of developing diabetes. Further, reducing FFAs levels should be an important goal in the management of patients with type 2 diabetes mellitus. Preventing obesity (BMI≥30kg/m2) could largely prevent diabetes. More large-scale clinical studies are required to understand the molecular mechanisms behind obesity causing insulin-resistance and the genetic relationship between both the diseases in order to develop better therapeutic remedies. Acknowledgements None. 13. Kalra S, Unnikrishnan AG. Obesity in India: The weight of the nation. J Med Nutr Nutraceut. 2012;1:37–41. 14. Yajnik CS, Ganpule-Rao AV. The obesity-diabetes association: What is different in Indians? Int J Low Extrem Wounds. 2010;9:113–115. 15. Seeley RJ, Woods SC. Monitoring of stored and available fuel by the CNS: Implications for obesity. Nat Rev Neurosci. 2003;4:901–909. 16. Spiegelman BM, Flier JS. Obesity and the regulation of energy balance. Cell. 2001;104:531–543. 17. Evans RM, Barish GD, Wang YX. PPARs and the complex journey to obesity. Nat Med. 2004;10:355–361. 18. Hutley L, Prins JB. Fat as an endocrine organ: relationship to the metabolic syndrome. Am J Med Sci. 2005;330(6):280–289. 19. Hussain A, Hydrie MZI, Claussen B, et al. Type 2 Diabetes and obesity: A review. Journal of Diabetology. 2010;1(2):4. 20. Redinger RN. The Pathophysiology of Obesity and Its Clinical Manifestations. Gastroenterol Hepat. 2007;3(11):856–63. 21. Straczkowski M, Kowalska I, Stepien A, et al. Increased plasmasoluble tumor necrosis factor alpha receptor 2 level in lean nondiabetic offspring of type 2 diabetic subjects. Diabetes Care. 2002;25(10):1824–1828. 22. Wellen KE, Hotamisligil GS. Inflammation, stress, and diabetes. J Clin Invest. 2005;115:1111–1119. Conflict of interest The authors declare there is no conflict of interest. References 157 1. Raval A, Dhanaraj E, Bhansali A, et al. Prevalence & determinants of depression in type 2 diabetes patients in a tertiary care centre. Indian J Med Res. 2010;132:195–200. 23. Verma S, Li SH, Wang CH, et al. Resistin promotes endothelial cell activation: further evidence of adipokine-endothelial interaction. Circulation. 2003;108(6):736–740. 2. American Diabetes Association. Diagnosis and Classification of Diabetes Mellitus, American Diabetes Association. Diabetes Care. 2010;33(Suppl 1):S62–S69. 24. Meigs JB, Wilson PW, Fox CS, et al. Body mass index, metabolic syndrome, and risk of type 2 diabetes or cardiovascular disease. J Clin Endocrinol Metab. 2006;91(8):2906–2912. 3. Misra A, Pandey RM, Devi J R, et al. High prevalence of diabetes, obesity and dyslipidemia in urban slum population in northern India. Int obes Relat Metab Disord. 2001;25:1722–1729. 25. 4. Mishra A. Khurana L. obesity and metabolic syndrome in developing countries. J Clin Endocriol Metab. 2008;93:59–30. Nagaya T, Yoshida H, Takahashi H, et al. Increases in body mass index, even within non-obese levels, raise the risk for Type 2 diabetes mellitus: a follow-up study in a Japanese population. Diabet Med. 2005;22(8):1107–1111. 26. 5. Halpern A, Mancini MC. Diabesity: are weight loss medications effective? Treat Endocrinol. 2005;4:65–74. Oguma Y, Sesso HD, Paffenbarger RS, et al. Weight change and risk of developing type 2 diabetes. Obes Res. 2005;13(5):945–951. 27. Wannamethee SG, Shaper AG, Walker M. Overweight and obesity and weight change in middle aged men: impact on cardiovascular disease and diabetes. J Epidemiol Community Health. 2005;59(2):134–139. 6. Stumvoll M, Goldstein BJ, van Haeften TW. Type 2 diabetes: principles of pathogenesis and therapy. Lancet. 2005;365:1333–1346. 7. Eckel RH, Kahn SE, Ferrannini E, et al. Obesity and Type 2 Diabetes: What Can Be Unified and What Needs to Be Individualized? Diabetes Care. 2011;34:1424–1430. 28. de Ferranti S, Mozaffarian D. The perfect storm: obesity, adipocyte dysfunction, and metabolic consequences. Clin Chem. 2008;54(6):945–955. 8. St- Pierre J, Lemieux I, Vohl MC, et al. Contribution of abdominal obesity and hyper-triglyceridesmia to impaired fasting glucose and coronary artery disease. A J Cardiol. 2002;90:15–18. 29. 9. Tataranni PA. Pathophysiology of obesity-induced insulin resistance and type 2 diabetes mellitus. Eur Rev Med Pharmacol Sci. 2002;6:27– 32. Weyer C, Funahashi T, Tanaka S, et al. Hypoadiponectinemia in Obesity and Type 2 Diabetes: Close Association with Insulin Resistance and Hyperinsulinemia. J Clin Endocrinol Metab. 2001;86(5):1930–1935. 30. Deng Y, Scherer PE. Adipokines as novel biomarkers and regulators of the metabolic syndrome. Ann N Y Acad Sci. 2010;1212:E1–E19. 10. Pratley RE, Weyer C, Bogardus C. Metabolic abnormalities in the development of noninsulin-dependent diabetes mellitus. In: LeRoith D, Taylor SI, et al. editors. Diabetes mellitus. Philadelphia: LippincotRaven Publishers;2000:548–557. 31. Pandya H, Lakhani JD, Patel N. Obesity is becoming synonym for diabetes in rural areas of India also – an alarming Situation. Int J Biol Med Res. 2011;2(2):556–560. 32. 11. Adams TD, Gress RE, Smith SC, et al. Long-term mortality after gastric bypass surgery. Engl J Med. 2007;357:753–761. Chan JM, Stampfer MJ, Ribb EB, et al. Obesity, fat distribution and weight gain as risk factors for clinical diabetes in man. Diabetes Care. 1994;17(9):961–969. 12. Buchwald H, Estok R, Fahrbach K, et al. Weight and type 2 diabetes after bariatric surgery: systematic review and metaanalysis. Am J Med. 2009;122:248–256. 33. Colditz GA, Willett WC, Rotnitzky A, et al. Weight gain as a risk factor for clinical diabetes mellitus in women. Ann Intern Med. 1995;122(7):481–486. Citation: Siddiqui S. Obesity and diabetes: interrelationship. Adv Obes Weight Manag Control. 2018;8(2):155‒158. DOI: 10.15406/aowmc.2018.08.00233 Copyright: ©2018 Siddiqui Obesity and diabetes: interrelationship 34. Snehalatha C, Vishwanathan V, Ramachandran A. Cutoff values for normal anthropometric variables in Asian Indian adults. Diab Care. 2003;26(5):1380–1384. 35. Everhart JE, Pettitt DJ, Bennett PH, et al. Duration of Obesity Increases the Incidence of NIDDM. Diabetes. 1992;41(2):235–240. 36. The Emerging Risk Factor Collaboration. Separate and combined associations of body-mass index and abdominal adiposity with cardiovascular disease: collaborative analysis of 58 prospective studies. Lancet. 2011;377(9771):1085–1095. 37. Montague CT, O’ Rahilly S. The perils of portliness: causes and consequences of visceral adiposity. Diabetes. 2000;49(6):883–888. 38. Rampersaud E, Damcott CM, Fu M, et al. Identification of novel candidate genes for type 2 diabetes from a genome-wide association scan in the Old Order Amish: evidence for replication from diabetesrelated quantitative traits and from independent populations. Diabetes. 2007;56(12):3053–3062. 39. Hayes MG, Pluzhnikov A, Miyake K, et al. Identification of type 2 diabetes genes in Mexican Americans through genomewide association studies. Diabetes. 2007;56:3033–3044. 158 44. Loos RJ, Bouchard C. Obesity–is it a genetic disorder? J Int Med. 2003;254:401–425. 45. Elbers CC, Onland-Moret NC, Franke L, et al. A strategy to search for common obesity and type 2 diabetes genes. Trends Endocrinol Metab. 2007;18(1):19–26. 46. HU H, LI X. Networking pathways unveils association between obesity and non-insulin dependent diabetes mellitus. Pac Symp Biocomput. 2008;13:255–266. 47. Statnick MA, Beavers LS, Conner LJ, et al. Decreased expression of apM1 in omental and subcutaneous adipose tissue of humans with type 2 diabetes. International Journal of Reproduction and Diabetes Research. 2000;1(2):151–158. 48. Matsuzawa Y. The metabolic syndrome and adipocytokines. FEBS. 2006;580(12):2917–2921. 49. Diez JJ, Iglesias P. The role of novel adipocyte-derived hormone adiponectin in human disease. Eur J Endocrinol. 2003;148(3):293– 300. 50. Hussain A, Hydrie MZI, Claussen B, et al. Type 2 Diabetes and obesity: A review. Journal of Diabetology. 2010;2:1. 51. Ouchi N, Ohishi M, Kihara S, et al. Association of hypoadiponectinemia with impaired vasoreactivity. Hypertension. 2003;42(3):231–232. 40. Scherag A, Dina C, Hinney A, et al. Two new loci for body-weight regulation identified in a joint analysis of genome-wide association studies for early-onset extreme obesity in French and German study groups. PLoS Genet. 2010;6(4):e1000916. 41. Lindgren CM, Heid IM, Randall JC, et al. Genome-wide association scan meta-analysis identifies three loci influencing adiposity and fat distribution. PLoS Genet. 2009;5(7):1. 52. Hotta K, Funahashi T, Arita Y, et al. Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients. Arterioscler Thromb Vasc Biol. 2000;20(6):1595–1599. 42. Bogardus C. Missing heritability and GWAS utility. Obesity (Silver Spring). 2009;17(2):209–210. 53. 43. Lander ES. Initial impact of the sequencing of the human genome. Nature. 2011;470(7333):187–197. Mohan V, Deepa R, Pradeepa R, et al. Association of low adiponectin levels with the metabolic syndrome–the Chennai Urban Rural Epidemiology Study (CURES–4). Metabolism. 2005;54(4):476–481. Citation: Siddiqui S. Obesity and diabetes: interrelationship. Adv Obes Weight Manag Control. 2018;8(2):155‒158. DOI: 10.15406/aowmc.2018.08.00233 

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MUDALIGE CHAMIKA GAJANAYAKA JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD LAWS 522: The Judiciary Submitted for the LLB(Honours) Degree Faculty of Law 2014 3 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD Table of Contents I Introduction ............................................................................................... 7 II The Saxmere Saga ..................................................................................... 9 III Judicial Recusal in New Zealand: The Current Paradigm ...................... 11 A B IV B VI 1 The test for apparent bias ....................................................... 11 2 Applying the test for apparent bias ......................................... 12 Recusal Procedure ........................................................................... 14 1 Disclosing an interest ............................................................. 15 2 How is a recusal motion made? .............................................. 15 3 How is a recusal motion dealt with? ....................................... 16 The Public Confidence Imperative ........................................................... 17 A V The Substance of Apparent Bias ....................................................... 11 Who Is The Public? .......................................................................... 19 1 The judiciary: an instrument ................................................... 19 2 The judiciary: a service provider ............................................ 20 3 The public: generally .............................................................. 21 What Fosters the Public's Confidence in the Judiciary? ................... 21 1 Dispute settlement from a judge's perspective ......................... 22 2 Dispute settlement from the public's perspective ..................... 22 Adjudication: A Process-based Perspective .............................................. 23 A Litigants Initiating Disputes ............................................................. 24 B An Adversarial System of Presenting Disputes ................................. 25 C Reasoned Decision-making .............................................................. 26 D Recourse to the Law......................................................................... 27 E An Impartial Arbiter ........................................................................ 27 F Process-based Adjudication: Generally ........................................... 29 Judicial Recusal: A Mismatch between Theory and Practice .................. 29 A Impediments to Litigants Initiating Recusal Motions ........................ 30 B A Lack of Adversarial Procedure ..................................................... 30 C A Dearth of Reasoned Decisions and Precedent ............................... 31 D The Archetypical Interested Arbiter ................................................. 31 E The Shortcomings of Current Judicial Recusal Procedure................ 32 VII A Way Forward: Procedural Reform ....................................................... 32 4 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD A Disclosure and Filing Recusal Motions ............................................ 33 1 Disclosure .............................................................................. 33 2 Process of questioning an interest and filing a recusal motion 34 B An Impartial Arbiter ........................................................................ 35 C Positive Externalities of an Independent Judge: Adversary; Reasoned Decisions; and Precedent ....................................................... 37 1 Adversarial process ................................................................ 37 2 Reasoned decisions generating precedent ............................... 37 VIII Procedural Reform: A Critique ................................................................ 37 A B IX Concerns with Transferring Recusal Motions to an Independent Judge ...................................................................................... 37 1 Practical concerns .................................................................. 37 2 Principled concerns ................................................................ 38 Alterations to the Proposed Reform ................................................. 39 1 When should a recusal motion be transferred to an independent judge?............................................................................ 39 2 Procedural posturing .............................................................. 40 3 Peremptory recusal................................................................. 40 C Focus on Substance Rather than Procedure ..................................... 41 D Reform: A Solution in Search of a Problem? .................................... 43 Saxmere Revisited, Following Reform ..................................................... 43 A Disclosure ....................................................................................... 44 B Filing a Recusal Motion ................................................................... 45 C An Independent Judge and its Corollary Benefits ............................. 45 X Conclusion ............................................................................................... 47 XI Bibliography............................................................................................. 49 5 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD Abstract The well-documented Wool Board Disestablishment Co v Saxmere Co litigation thrust the often dormant issue of conflicts of interest between a judge and a litigant into the limelight. Now that the dust has settled on the controversy that culminated in Wilson J's resignation, it is pertinent to question the status quo and investigate the potential cause of the events and, more importantly, whether measures need to be taken to prevent another such occurrence. This paper takes a principled approach to analysing judicial recusal law in New Zealand, with a particular focus on procedure. In doing so, a mismatch between process theory and the reality of haphazard self-regulation highlights the procedural shortcomings of the current recusal paradigm. To remedy this, the author applies aspects of process theory to reform judicial recusal procedure and bring it in line with general civil litigation practice. The proposed reform instils some fundamental practices that are presently absent in recusal procedure. To contextualise the paper's findings, the author revisits the Saxmere saga to first posit that a lack of procedural safeguards may have contributed to the saga and two, to suggest that had the procedural safeguards proposed by this paper been in place, the controversy could have been mitigated, if not avoided. Judicial Recusal – Procedural Reform – Process Word Count The text of this paper (excluding title page, table of contents, abstract, footnotes and bibliography) comprises approximately 14,400 words. 7 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD I Introduction "Whoever has the legislative or supreme power in any commonwealth … is bound to govern by established standing laws, promulgated and known to the people (and not by on-the-spot decrees), with unbiased and upright judges appointed to apply those laws in deciding controversies."1 John Locke's seventeenth century articulation of the need to resolve "legal disputes by impartial and independent judges is recognised as an essential underpinning of western society". 2 The United Nations' Universal Declaration of Human Rights affirms that "everyone is entitled in full equality to a fair and public hearing by an independent and impartial tribunal". 3 In New Zealand, an independent arbiter is a fundamental freedom affirmed by the Bill of Rights Act 1990. 4 Judges are reminded of the importance of adjudicating impartially upon entering the bench. The judicial oath proclaims that judges must "do right to all manner of people after the laws and usages of New Zealand without fear or favour, affection or ill will". 5 To be impartial, one must be detached and disinterested so that one’s personal views and interests do not weigh in on the matter at hand. Recusal is the means by which judicial impartiality is addressed and maintained.6 In accordance with Lord Hewart CJ's well-cited dictum that "it is not merely of some importance but is of fundamental importance that justice should not only be done, but should manifestly and undoubtedly be seen to be done",7 judges must not only be impartial, they must appear to be impartial. If a judge does not – or notably, if a judge appears to not – bring an impartial mind to adjudicating a dispute, litigants may endeavour to have the judge recused. Alternatively, a judge may recuse themselves sua sponte if they acknowledge that their impartiality may be, or may appear to be, compromised. While easily preached and often assumed, judicial impartiality, and in particular the method of ensuring it, can be difficult to understand. 1 John Locke Second Treatise of Government (Awnsham Churchill, 1690). Muir v Commissioner of Inland Revenue [2007] NZCA 334, [2007] 3 NZLR 495 at [32]. 3 The Universal Declaration of Human Rights, GA Res 217A, III UN GAOR A/810 (1948) 10. 4 Bill of Rights Act 1990, ss 25 and 27. Section 25(a) affirms the right to a fair and public hearing by an independent and impartial court in matters of criminal law. Section 27 affirms the right to observe the principles of natural justice, which Hammond J finds to "undoubtedly [encompass] … the proposition that judges must be independent and impartial" in Muir v Commissioner of Inland Revenue, above n 2, at [32]. 5 Oaths and Declarations Act 1957, s 18. 6 Throughout this paper, judicial recusal and judicial disqualification will be conflated and used interchangeably. Strictly speaking, there is a technical distinction between the two terms; disqualification usually reflects a legal requirement for a judge to not sit on a case, for instance due to a per se rule of financial interest with a party, whereas recusal usually reflects a judge deciding to not sit on a case sua sponte. 7 R v Sussex Justices, ex parte McCarthy [1924] 1 KB 256 at 259. 2 8 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD As Professor Charles Geyh observed, "the legal establishment is deeply divided over when it is reasonable for the presumption of impartiality to yield to the suspicion that extra-legal influences may have compromised the judge's impartial judgment".8 This paper critiques judicial recusal law in New Zealand.9 Its analysis also has international application as some of the substantive law and many of the processes are prevalent in other common law jurisdictions. New Zealand's current Chief Justice, Sian Elias CJ, articulated in a recent speech that "today respect for courts – without which the rule of law is in trouble – has to be earned and re-earned by doing and the doing has to be painstakingly courteous and reasoned". 10 The Chief Justice's remarks provide some relevance to this paper's inquiry by posing the question: is judicial recusal law effective in earning the public's respect for New Zealand's courts? The significance of this paper's inquiry is heightened by recent events that culminated in New Zealand's first and only judicial resignation amidst allegations of an appearance of partiality; the resignation of Wilson J (the Saxmere saga). The author hypothesises that the current recusal paradigm is inadequate to fulfil its principle role of helping to maintain an appearance of judicial impartiality. Further, the author opines that an absence of procedural safeguards may have contributed to the Saxmere saga. And conversely, that had procedural safeguards been in place, the controversy may have been mitigated, if not avoided. This paper will first summarise the well-documented Saxmere saga to contextualise the ensuing discussion of law, principles and reform. The critique begins with an overview of the substantive law and procedure of judicial recusal in New Zealand. Thereafter, this paper will turn to first principles in looking at how the judiciary is legitimatised. In doing so, public confidence is found to be one of the key legitimising traits of the judiciary with procedural fairness being a primary driver of the public's confidence. The main adjudicatory practices that bolster the public's perceptions of procedural fairness will then be analysed in New Zealand's civil litigation environment. 8 Charles G Geyh "Why Judicial Disqualification Matters. Again" (2011) 30 The Review of Litigation 671 at 676. 9 The paper focuses its analysis on the High Court and the two appellate courts in New Zealand, the Court of Appeal and the Supreme Court. Further, the paper concentrates on judicial recusal at first instance, rather than on substantive appeals to higher courts based on grounds of partiality or an incorrectly adjudicated recusal motion. Research from overseas jurisdiction, such as the United States, are referred to at times to provide some psychological research and examples. While acknowledging that the law in New Zealand materially differs from that in the United States, the underlying procedures relating to judicial recusal are similar. This is also a reflection on the relative dearth of New Zealand-centric scholarship on judicial recusal. 10 Sian Elias, Chief Justice of New Zealand "Contemporary Issues for Courts – Demystifying the Judicial Process" (Conference of Chief Justices of Asia and Pacific 2013, Supreme Court, Singapore, 29 October, 2013). 9 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD Existing judicial recusal procedures will be compared and contrasted with best-practice adjudication to find that they are deficient and out of line with civil litigation practices in New Zealand. This paper then proposes a number of principled procedural reforms to realign recusal with the civil litigation process. The proposals will be critiqued before finally returning to the Saxmere saga to highlight some of the procedural shortcomings that may have caused the controversy and to personify the proposed reform by hypothetically applying the procedural reform to the Saxmere saga. In doing so, the author postulates that procedural reform holds considerable promise as the way forward for judicial recusal in New Zealand and indeed other common law jurisdictions. II The Saxmere Saga The events that culminated in Wilson J's resignation are summarised below. 11 They are intended to provide some context for the analysis to follow before being revisited in Part IX to compare and contrast New Zealand's current judicial recusal law with this paper's proposed procedural reforms. Justice Wilson, 12 then a Judge of the Court of Appeal, was appointed to sit on a case between the Wool Board and Saxmere.13 Counsel for the appellant, the Wool Board, was Alan Galbraith QC. Justice Wilson and Galbraith were good friends from their time practicing together at the bar. The two were also business partners in a horse stud, Rich Hill. 14 In acknowledging the materiality of his business relationship with Galbraith, Wilson J privately telephoned opposing counsel, Francis Cooke QC, to inform him of their mutual business interest. The details of Wilson J's disclosure to Cooke is unclear but it seems to have been distinctly general. 15 On the basis of the information Wilson J disclosed in the phone call and after consulting with his instructing solicitors, Cooke informed the Registrar of the court that the respondents had no objection to Wilson J 11 See: Judicial Conduct Commissioner Decision of the Judicial Conduct Commissioner as to Three Complaints Concerning Justice Wilson (7 May 2010) at [34] for a detailed account of the events. 12 WM Wilson QC is no longer a Judge of the High Court of New Zealand following his resignation from office in 2010. However, for ease, he will be referred to as Wilson J in this paper. 13 Wool Board Disestablishment Co v Saxmere Co [2007] NZCA 349, [2007] BCL 885 [Saxmere No 1]. For ease, the respondents, which include four parties, will be referred to as Saxmere throughout the paper. 14 Gerard McCoy "Judicial Recusal in New Zealand" in HP Lee Judiciaries in Comparative Perspective (Cambridge University Press, 2011) 322 at 330: "[Rich Hill] was a really significant enterprise with revenue in the millions of dollars and it owned livestock and land". Nicky Hager "Appeal Court Judge Faces a Question of Judgement" The Sunday Star Times (New Zealand, 24 August 2008): the business is "worth tens of millions of dollars". 15 Saxmere Co v Wool Board Disestablishment Co [2009] NZSC 72, [2010] 1 NZLR 35 at [17] [Saxmere No 2]. Judicial Conduct Commissioner, above n 11, at [34]. 10 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD sitting on the appeal. The case duly proceeded and the court upheld the Wool Board's appeal. Subsequent to the case, Saxmere discovered that the business relationship between Wilson J and Galbraith was substantially greater that it had thought. On the basis that Wilson J had not disclosed the extent of his business relationship with Galbraith, Saxmere appealed to the Supreme Court to have the Court of Appeal's judgment set aside.16 Prior to the hearing of the appeal, Wilson J accepted an invitation to further disclose the nature of his business relationship with Galbraith to the Supreme Court. In hearing the appeal, the court concluded that there was no evidence that Wilson J was "beholden to Mr Galbraith because of the business dimension of their relationship". 17 Thus, the business relationship was found not to give rise to a reasonable apprehension of bias and the appeal was dismissed. The Supreme Court's finding of no evidence that Wilson J had been indebted or otherwise beholden to Galbraith turned out to be crucial. 18 Saxmere applied to the Supreme Court to recall its judgment on the basis that key points regarding the business relationship had been overlooked or not disclosed.19 Wilson J responded to the Supreme Court's request for further disclosure. It transpired that Wilson J had omitted several key facts that had led to the Supreme Court assessing Rich Hill as a "passive"20 company that Wilson J and Galbraith made "equal contributions"21 to. In fact, when Saxmere No 1 was heard in the Court of Appeal, Wilson J was indebted to Galbraith due to unequal contributions by the shareholders in anticipation of an investment by the company. 22 In light of these new facts, the Supreme Court recalled its earlier decision and allowed Saxmere's appeal to set aside the Court of Appeal's judgment and undertake a retrial. Following several complaints to the Judicial Conduct Commissioner, a Judicial Conduct Panel was appointed to investigate Wilson J's conduct throughout the aforementioned events. Before the Judicial Conduct Panel could deliver its decision, Wilson J resigned. Writing on the saga, Gerard McCoy stated that the Supreme Court's "cachet and prestige … must have been inadvertently wounded"23 as a result of the controversy and resignation. This paper does not endeavour to affirm or negate the 16 Saxmere No 2, above n 15. Saxmere No 2, above n 15, at [25]. 18 B Robertson "Judicial Ethics" [2010] 121(4) NZLJ 121 at 121. 19 Saxmere Co v Wool Board Disestablishment Co [2009] NZSC 122, [2010] 1 NZLR 76 [Saxmere No 3]. 20 Saxmere No 3, above n 15, at [15]. 21 Saxmere No 3, above n 15, at [15]. 22 Saxmere No 3, above n 15, at [15]–[16]. Wilson J was indebted to Galbraith by an aggregate sum of over $242,000. 23 McCoy, above n 14, at 335. 17 11 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD accuracy of McCoy's observations. Rather, it is concerned with the law and procedure that left open the possibility for a highly-regarded legal mind to conduct himself in this manner. In Part IX, the Saxmere saga will be revisited to test the author's hypothesis that: one, a lack of procedural safeguards may have contributed to the controversy; and two, that had the procedural reform that is to be proposed in this paper been in place, the controversy could have been mitigated, if not avoided. III Judicial Recusal in New Zealand: The Current Paradigm In New Zealand, there is no legislative oversight for assessing the potentially disqualifying nature of a judge's interest with a party or counsel. In this regard, the judiciary is self-regulated. Grant Hammond24 described the current framework as: "perched in something of a no-man's land between statutes and common law".25 Without looking to confine discussion to particular types of interests that may bring a judge's impartiality into question, this paper will consider apparent bias generally as the catalyst for recusal sua sponte or as the basis for filing a recusal motion by a party. 26 This Part will set out the test for assessing apparent bias and the procedure for making and handling recusal motions. A The Substance of Apparent Bias 1 The test for apparent bias Prior to 2007, the law for establishing apparent bias was "in an awkward state in New Zealand". 27 This was due in part to earlier conflicting approaches to apparent bias in the United Kingdom and Australia.28 Now, notwithstanding "some semantic differences, the test [for apparent bias] in the United Kingdom and Australia have become essentially the In this paper, Grant Hammond is cited both judicially – when he is sitting as a justice of the Court of Appeal – and when he is writing extra-judicially. He will be referred to accordingly. 25 Grant Hammond Judicial Recusal: Principles, Process and Problems (Hart Publishing, Portland, 2009) at 154. 26 Hammond, above n 25, at 15. Traditionally, bias has been categorised into actual bias and apparent bias. Actual bias dealt with a situation where a judge subjectively had "regard to something other than the true merits of the dispute". On the other hand, apparent bias dealt with a situation where it objectively appeared that a judge may have "regard to something other than the true merits of the dispute". The burden of subjectivity required for actual bias made it notoriously difficult, and in fact unnecessary, to establish. Consequently, litigants and courts have focused on apparent bias as the primary situation when a judge should not sit on a case. 27 Muir v Commissioner of Inland Revenue, above n 2, at [44]. 28 The approach in the United Kingdom in the late twentieth century was articulated by Lord Goff in R v Gough [1993] AC 646 at 670, with the question being: "whether there was a real danger of bias on the part of [the judge]". A year later, Deane J in the High Court of Australia in Webb v R (1994) 181 CLR 41 at 70 stated that the "House of Lords test differs from that accepted in recent cases in this Court" and maintaining that "the fair-minded lay observer" is the standard to be applied in Australia. 24 12 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD same".29 The Supreme Court in Saxmere No 2 affirmed the Court of Appeal's approach in Muir to bring New Zealand law in line with the United Kingdom and Australia. 30 The Supreme Court set out that a judge should recuse themselves: 31 "if a fair-minded lay observer might reasonably apprehend that the judge might not bring an impartial mind to the resolution of the question the judge is required to decide". A two stage inquiry is required. 32 First, "establish the actual circumstances which have a direct bearing on a suggestion that the judge was or may be seen to be biased". 33 The factual inquiry must be "rigorous"34 to avoid frivolous or unsubstantiated claims of bias. Second, the fair-minded lay observer is considered in determining whether it would "reasonably apprehend that the judge might not bring an impartial mind to the resolution of the case".35 The High Court of Australia emphasised that this "question is one of possibility (real and not remote), not probability" 36 and that "no attempt need be made to inquire into the actual thought processes of the judge". 37 Nor should there be any consideration as to whether another judge may be better suited to hear a case; the question is wholly directed at the impartiality of the judge in question.38 2 Applying the test for apparent bias While there is a wide range of interests that could affect a fair-minded lay observer's perception of a judge's impartiality, Dean J articulated "four distinct, though sometimes overlapping, main categories of [interests that may raise a reasonable apprehension of bias]":39  having an interest, including a pecuniary and non-pecuniary interest, in a person or entity that is also interested in the case; 29 Saxmere No 2, above n 15, at [3]. In Australia, Ebner v Official Trustee in Bankruptcy (2000) 205 CLR 337 at [6]: the majority of the court set out the test as whether a: "fair-minded lay observer might reasonably apprehend that the judge might not bring an impartial mind to the resolution of the question the judge is required to decide". In the United Kingdom, Porter v Magill [2002] 2 AC 357 at [103] Lord Hope asks: "whether the fair-minded and informed observer, having considered the facts, would conclude that there was a real possibility that the tribunal was biased." 30 Muir v Commissioner of Inland Revenue, above n 2, at [60]–[64]. 31 Saxmere No 2, above n 15, at [3]. 32 Muir v Commissioner of Inland Revenue, above n 2, at [62]. 33 Muir v Commissioner of Inland Revenue, above n 2, at [62]. 34 Muir v Commissioner of Inland Revenue, above n 2, at [62]. 35 Muir v Commissioner of Inland Revenue, above n 2, at [62]. 36 Ebner v Official Trustee in Bankruptcy (2000) 205 CLR 337 at [7]. 37 Ebner v Official Trustee in Bankruptcy (2000) 205 CLR 337 at [7]. 38 Aussie Airlines Pty Ltd v Australian Airlines Pty Ltd [1996] 65 FCR 215, (2006) 135 ALR 753 at 230– 231. 39 Webb v R, above n 28, at 74. 13 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD  engaging in conduct that leads to an apprehension of bias, either in the course of, or outside the proceedings;  being associated, either directly or indirectly, with a person or persons interested in the proceedings; and  obtaining extraneous information, namely prejudicial knowledge that may give rise to an apprehension of bias. Key to the apparent bias test is the mind of the fair-minded lay observer. In Saxmere No 2, Blanchard J presumed the observer to be: "intelligent and to view matters objectively"; a "non-lawyer but [be] reasonably informed about the workings of our [New Zealand] judicial system";40 and "neither unduly sensitive or suspicious nor complacent about what may influence the judge's decision". 41 In the House of Lords, Lord Hope classified the observer as one who always reserves judgement and justifies any conclusions objectively. 42 Commentators have viewed the observer as "a paragon of virtue and so unlike the average member of the public."43 It is clear that the indeterminacy of applying the fair-minded lay observer can lead reasonable minds to differ when applying the test to a judge's potentially conflicting interest. When considering a recusal issue, judges must also discharge their duty to sit and consider the doctrine of necessity. (a) Duty to sit The judiciary's Guidelines for Judicial Conduct sets out that "judges have an obligation to sit on any case allocated [to them]"44 and "should not accede too readily to suggestions of bias". 45 Justice Hammond described the duty to sit as a "counterbalance" 46 that "helps protect judicial independence". 47 Former Australian Chief Justice Mason CJ more thoroughly emphasised the duty to sit:48 "it is equally important that judicial officers discharge their duty to sit and do not, by acceding too readily to suggestions of appearance of bias, encourage parties 40 Saxmere No 2, above n 15, at [5]. Saxmere No 2, above n 15, at [5]. 42 Helow v Secretary of State for the Home Department [2008] 1 WLR 2416 at [1]–[3]. 43 Philip Havers and Alasdair Henderson "Recent Developments (And Problems) In the Law On Bias" (Hart Judicial Review Conference, The Cavendish Conference Centre, London, 10 December 2010) at [9]. Havers and Henderson provide a succinct overview of the fair-minded lay observer at [8]–[18]. 44 "Guidelines for Judicial Conduct" (March 2013) Courts of New Zealand <www.courtsofnz.govt.nz> at [27]. 45 "Guidelines for Judicial Conduct", above n 44, at [29]. 46 Muir v Commissioner of Inland Revenue, above n 2, at [35]. 47 Muir v Commissioner of Inland Revenue, above n 2, at [35]. 48 Re JRL; ex parte CJL (1986) 161 CLR 342 at 352. Chief Justice Mason is referring to "judge-shopping" here which is the idea that litigants may seek to have their case heard by a particular judge on the basis that they may adjudicate more favourably to them. 41 14 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD to believe that by seeking the disqualification of a judge, they will have their case tried by someone thought to be more likely to decide the case in their favour." An apolitical judicial appointment process and relatively neutral extrajudicial utterances suggest that "judge-shopping" is not as live an issue in New Zealand as it may be in other jurisdictions. Nonetheless, the weight judges give to their duty to sit is not universal. Geyh observed that "judges of a more traditionalist bent will guard the presumption of impartiality far more zealously than those with … more 'realist' learnings".49 Traditionalistic views are a hangover from William Blackstone's near irrefutable presumption of judicial impartiality based on the idea that judges could strictly follow the law whilst disassociating themselves from any extra-legal influences.50 The realist approach more readily acknowledges that judges may be subject to extra-legal pressures. Additionally, the duty to sit may be stronger in appellate courts due to the limited number of judges that may be available to replace a judge if they decide not to sit. if a judge recuses themselves. (b) Necessity There may be instances where, in the interests of justice, a judge has to sit on a case even when doing so would invoke an appearance of partiality in the mind of a fair-minded lay observer. The necessity doctrine is required on the basis that litigation cannot be non sequitur.51 Circumstances where necessity may be invoked are limited. The classical example is where a matter concerns the judiciary as a whole, such as questions of judicial remuneration. In New Zealand, the necessity doctrine is almost non-existent because its appellate courts, the Court of Appeal and Supreme Court, have robust procedures to call upon judges temporarily when a full quorum comprising their permanent judges is not possible.52 B Recusal Procedure In Muir, Hammond J remarked that: "there is little discussion of this [judicial recusal procedure] issue in the cases, but there are some real difficulties in this area". In fact, he 49 Geyh, above n 8, at 698. Geyh, above n 8, at 678, n 27. 51 Hammond, above n 25, at 99: "there cannot be a litigation system in which it is impossible to litigate a given case". 52 Quorum constitution is primarily an issue in a jurisdiction's highest court because a full court is usually required to sit on a case. If a judge is to not sit, then the quorum will not be fully constituted and jurisdictional issues arise. The issue is not prevalent in New Zealand's highest court, the Supreme Court, because temporary judges can be asked to sit on cases where a permanent member cannot sit. Supreme Court Act 2003, s 23. Section 23(1) states: "the Governor-General may appoint as acting Judges of the Supreme Court retired Judges of the Supreme Court or the Court of Appeal". 50 15 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD conceded extrajudicially that "it is difficult to review the procedural law in the recusal area because by and large there is none." 53 Judicial self-regulation has led to a distinctly informal and ad hoc basis for parties to question a judge's impartiality. 1 Disclosing an interest Clearly, parties require knowledge of a judge's potentially disqualifying interest before a recusal motion can be made. Otherwise, parties may not be aware of, nor may they have cause to inquire into, a judge's interests. The Judicial Conduct Guidelines prescribe that judges should make "as early as possible … in writing and through the Registrar of the Court to counsel for all parties". 54 The level of disclosure "should ensure that the parties have sufficient information, without unnecessary detail, to decide whether to make a recusal motion". 55 There may also be instances where a party is aware of intricacies regarding a disclosed interest that a judge is unaware of. If so, counsel should disclose the supplementary information through the Registrar of the court.56 Litigants are however not permitted to question a judge in search of potentially disqualifying interest; disclosure is a judge's duty and burden. As Blanchard J observed: 57 "it is for a judge who makes a disclosure to ensure that the parties have enough information, shorn of unnecessary detail, to make up their minds about whether to make a recusal application. They and their counsel should not be placed in the embarrassing position of having to seek further information from the judge." 2 How is a recusal motion made? The appropriate procedure for raising issues of partiality and filing a recusal motion is unclear. One commentator recognised that "there is no uniform practice as to the means by which an objection [to a judge sitting on a case] may be raised". 58 At first instance, a party question a judge about a potentially disqualifying interest in chambers with counsel for all parties present or in open court through the court's Registrar. 59 If the judge is not of the view that the interest warrants recusal and so continues to sit, a party may further pursue the matter by filing an originating application supported by affidavits. A judge 53 Hammond, above n 25, at 81. "Guidelines for Judicial Conduct", above n 44, at [42]. 55 "Guidelines for Judicial Conduct", above n 44, at [42]. 56 "Guidelines for Judicial Conduct", above n 44, at [42]; and "Court of Appeal Recusal Guidelines" (June 2013) Courts of New Zealand <www.courtsofnz.govt.nz> at 2(d). 57 Saxmere No 2, above n 15, at [34]. Litigants cannot "fish" for potentially disqualifying interests. 58 Melissa A Perry "Disqualification of Judges: Practice and Procedure – Discussion Paper" (Victoria, Australian Institute of Judicial Administration, 2001) at [2.29]. For an exposition of the varied circumstances that recusal motions may be made, see: Perry, above n 58, at [2.29]. 59 Hammond, above n 25, at 81. 54 16 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD will then be required to formally respond. Questions of partiality may arise at different stages of the proceedings. 60 If a party is fully aware of a judge's potentially disqualifying interest throughout a proceeding but fails to raise concerns about it or does not file an originating application in a timely manner, that party will be deemed to have provided fully informed consent for the judge to sit on the case and no subsequent appeals can be made on the basis of partiality. In effect, this is an implied waiver by parties to forego considerations of an appearance of bias. 61 3 How is a recusal motion dealt with? The practice amongst the majority of common law jurisdictions, including New Zealand, is for the challenged judge to determine recusal motions made against them.62 Judicial conduct guidelines reinforce this practice. In New Zealand, the Guidelines for Judicial Conduct state that "the question of disqualification is for the judge." 63 Similarly, the Australian Guide to Judicial Conduct states that "if a judge considers that disqualification is required, the judge should so decide" 64 and that "disqualification is for the judge to decide".65 Even in appellate courts where a panel of judges sit, the recusal decision remains with the challenged judge. Judges may consult a potentially conflicting interest with other judges. The Court of Appeal Recusal Guidelines state that "where the issue is not clear cut, the judge should consult … with other members on the".66 A judge may, in light of prior knowledge of an interest between themselves and the parties, choose to recuse themselves sua sponte even before being listed to sit on a case. The Court of Appeal Recusal Guidelines recognise the convenience of prophylactic recusals by allowing a judge to "indicate cases on which they should not be listed, for conflict reasons". 67 However, ex ante recusals can be impractical in trial courts where judges may be listed to sit on a case with short notice. In the Court of Appeal and the Supreme Court, it is more tenable for a judge to recuse themselves prior to being listed on a case due to sufficient temporal gaps between the filing and hearing of a case. 60 Perry, above n 58, at [1.14]. Perry sets out different stages of the proceedings at which a recusal issue may arise: "at the interlocutory stage, during the course of the trial, during summing up to a jury or in the course of sentencing remarks, after judgment has been reserve, upon delivery of judgment by reason of statements in the judge's reasons, or on appeal." 61 Saxmere No 2, above n 15, at [36]. 62 Hammond, above n 1, at 148. For examples see: "Guidelines for Judicial Conduct", above n 44, at [29]; and "Court of Appeal Recusal Guidelines", above n 56. 63 "Guidelines for Judicial Conduct", above n 44, at [29]. 64 The Council of Chief Justices of Australia "Guide to Judicial Conduct" (March 2007) Supreme Court of Western Australia <www.supremecourt.wa.gov.au> at [3.5]. New Zealand's Chief Justice is a part of the Council of Chief Justices of Australia. 65 The Council of Chief Justices of Australia "Guide to Judicial Conduct" at [3.5]. 66 "Court of Appeal Recusal Guidelines", above n 56, at [2(c)]. 67 "Court of Appeal Recusal Guidelines", above n 56, at 2(a). 17 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD As alluded to, conflict of interest issues may be dealt with in chambers between the parties and the challenged judge. If an originating application is subsequently made, it should be dealt with immediately in open court to minimise any wasted litigation if a judge decides to recuse him or herself. The judge will announce his or her decision via a Minute. A judge is not required to provide reasons for upholding or dismissing an application. A judge's decision to dismiss a recusal motion can form the basis of an appeal against a judgment.68 Alternatively, a litigant may make a similar complaint to the Judicial Conduct Commissioner, whose decisions are subject to judicial review. 69 Justice Mellissa Perry of the Federal Court of Australia stated extra-judicially that the apparent ease by which parties can raise questions of a judge's impartiality "masks the complex and difficult issues which impact upon the adjectival aspects of disqualification in the courts".70 The informality generates a lack of clarity in judicial recusal procedure that is unique when compared to other aspects of the litigation process. The existing judicial recusal procedure will later be compared and contrasted to certain aspects of civil litigation procedure in New Zealand. IV The Public Confidence Imperative The judiciary is unlike the other two branches of government, the executive and the legislature, in that its legitimacy is not conditional upon the approval of a constituency. It is not exposed to the direct public accountability that the legislature and executive are subject to by way of an electorate’s democratic rights. The separation of powers doctrine also protects the judiciary from indirect public accountability to the legislature and executive by virtue of security of tenure71 and financial security.72 Without a constituent mandate or public accountability, the judiciary's institutional legitimacy must originate from elsewhere. Notwithstanding its lack of public accountability, Hammond identified public confidence as a source of the judiciary's legitimacy when he stated that:73 68 Hammond, above n 25, at 101. The Judicial Conduct Commissioner was established by the Judicial Conduct Commissioner and Judicial Conduct Panel Act 2004. The purpose of the Act, set out in s 4, was to "enhance public public confidence in, and to protect the impartiality and integrity of, the judicial system", s 4. In dealing with complaints, the Judicial Conduct Commissioner may: undertake no further action in respect of the complaint, s 15A; dismiss the complaint, s 16; refer the complaint to the Head of the Bench, s 17; or recommend that the judge complained of be removed, s 18. For example: Wilson v Attorney-General [2011] 1 NZLR 399; and Muir v Judicial Conduct Commissioner [2013] NZHC 3507. 70 Perry, above n 58, at [1.11]. 71 Constitution Act 1986, s 23. 72 Constitution Act 1986, s 24. 73 Hammond, above n 25, at 5. 69 18 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD "society rightly looks to the courts as bastions of the Rule of Law. If the public cannot look with confidence to judges … the very notion of a 'legal system' as a fundamental pillar of western society would collapse." Philip Joseph, a prominent New Zealand constitutional and public law commentator, recognised the importance of public confidence for the judiciary when he stressed that "justice must be rooted in confidence".74 Without public confidence, disgruntled members of society may attempt to resolve disputes themselves, which in turn could compromise the Rule of law.75 While this paper will focus on public confidence, it is not the only trait that legitimises the judiciary. Judicial independence from legislative, executive and public pressures is also recognised as an "unalterable" fundamental of the judiciary. 76 Joseph, in finding that "confidence is destroyed when right-minded people go away thinking 'the judge was biased',"77 concluded that "impartiality represents the ultimate value associated with adjudication." 78 Judges have also recognised the importance of impartiality. For example, when outlining the advantages of the apparent bias test in Saxmere No 2, McGrath J highlighted that that test "gives significant weight to the need for public confidence in the integrity of the judicial system".79 Thus, judicial impartiality – and what is more, the appearance of judicial impartiality – seems to be one of the primary means of promoting public confidence. 80 And so, judicial recusal law, with its purpose being to ensure that dispute are decided by impartial judges, as well as appearing to be decided by impartial judges, inherently serves to protect public confidence. In order to analyse whether the current judicial recusal paradigm is sufficiently robust to help legitimise the judiciary, it is pertinent to further articulate the public confidence imperative. To do so, this Part will address two fundamental aspects of public confidence: first, identify who the public is to understand whose confidence, or lack thereof, may affect the judiciary’s legitimacy; and second, how the public's confidence is attained and maintained. 74 Philip A Joseph Constitutional and Administrative Law in New Zealand (4th ed, Brookers, Wellington, 2014) at 797. Here, Joseph is quoting Metropolitan Properties Co (FGC) v Lannon [1969] 1 QB 577 (CA) at 599. 75 For example: Tom R Tyler "The Role of Perceived Injustice in Defendants' Evaluations of Their Courtroom Experience" (1984) 18(1) Law & Society Review 51 at 51–52: "a lack of public support leads to a willingness to disobey the law and to engage in anti-system behaviours such as riots." 76 Robin Cooke "Fundamentals" (1988) NZLJ 158 at 164. 77 Joseph, above n 74, at 797. Here, Joseph is quoting Metropolitan Properties Co (FGC) v Lannon [1969] 1 QB 577 (CA) at 599. 78 Joseph, above n 74, at 798. 79 Saxmere No 2, above n 15, at [92]. 80 Joseph, above n 74, at 797. 19 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD A Who Is The Public? In conceptualising whose confidence, or lack thereof, may affect the public's confidence in the judiciary, it is useful to consider whose confidence, or lack thereof, may not affect the public's confidence in the judiciary. Defining the public as the electorate would be misleading because, as alluded to earlier, the judiciary is not accountable to constituents in the way the executive and legislature are.81 Nor should the media represent the public because the judiciary is not answerable to the media; it does not respond to media criticism of its performance. 82 Other representative bodies, such as the New Zealand Law Society, are left to respond to any such criticism as they see fit. To conceptualise the public in either of these ways may inadvertently marginalise the Rule of law by encouraging the judiciary to rule according to popular public sentiment, rather than established legal rules. It is useful to look at the judiciary's functions as the basis for conceptualising who the public is. 1 The judiciary: an instrument The judiciary may be viewed as an instrument whose role is to maintain society's right to justice by adjudicating disputes in a just manner. It has been noted that the judiciary is not democratically accountable. Reflecting judicial independence, nor is the judiciary accountable to the executive and legislature. However, the prevailing lack of direct democratic or institutional accountability may not be static. If the public's confidence in the judiciary materially decreases due to a perception that the judiciary is not carrying out its instrumental purpose of maintaining society's right to justice, the public may attempt to influence the judiciary via the legislature.83 Subjecting the judiciary to politicking is a more prevalent concern in jurisdictions like the United States where the judicial appointment process is less apolitical and judges have the ability to strike down unconstitutional legislation. In New Zealand, judges are independently appointed and judicial power is more constrained, which suggests that the possibility of the legislature interfering with the judiciary is relatively low. In saying that, it is not inconceivable for the independence of the New Zealand judiciary to be marginalised following a loss of public confidence. For example, judicial sentencing is one area where political pressure on the judiciary is increasing. Recent efforts by bodies like the Sensible Sentencing Trust have sought to limit judicial discretion and increase legislative prescription in criminal sentencing due to public 81 Geyh, above n 8, at 721–727. Geyh, above n 8, at 721–727. 83 Geyh, above n 8, at 725. 82 20 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD sentiment that punitive sentences were not sufficiently deterring repeat offending. Its lobbying influenced a change in sentencing law with the enactment of the Sentencing and Parole Reform Act 2010.84 The notion that the judiciary’s instrumental role is to maintain societal justice is a strong one. Conceptualising the public from this perspective can lead to different definitions of the public. Geyh observed that from an instrumental perspective, the judiciary may primarily be concerned about the confidence of those that are "engaged enough to act upon its dissatisfaction [of the court's instrumental performance]". 85 While acknowledging that perhaps there is a minority in New Zealand that may look to the legislature to influence the judiciary, this paper opines that this an insignificant minority and that a significant portion of the public would be necessary to influence the judiciary via the legislature. In conceptualising the public from the judiciary's instrumental function, the author postulates a much wider definition of the public. This thought is based on the idea that societal justice affects all of society and so the judiciary's legitimacy may be affected by the confidence, or lack thereof, of any member of society, albeit to a greater or lesser extent, rather than simply those that are sufficiently engaged to actively act upon their dissatisfaction. Thus, the public from an instrumental perspective should be defined as a jurisdiction's whole society, both individually and collectively. 2 The judiciary: a service provider The judiciary may prima facie be viewed as a state-sponsored dispute resolution service to society. Naturally, the primary focus of any service provider is likely to be its customers and ensuring that they are satisfied with the service so that they will return for repeat custom. Adopting this notion, the "customers" of the judiciary are the litigants that appear before the court.86 Litigants are whom the bench and bar alike have direct influence over. However, confining the judiciary's customers and influence as narrowly as this may be misleading. Simon Shetreet stated that "at one time courts were viewed merely as a dispute resolution institution … [but] today it is widely recognised that the judiciary performs the function of law making."87 This suggests that in resolving disputes, the judiciary is undertaking a de facto law-making function. Per the Rule of law, law affects every member of society. Thus, if it can be said that the judiciary has a surrogate law-making function, notwithstanding it is subject to parliament's sovereignty, 84 Sentencing and Parole Reform Act 2010, s 3(b): the Act's purpose is to "impose maximum terms of imprisonment on persistent repeat offenders who continue to serious violent offences." The Act's primary change was to enact the "three-strikes" law for repeat offenders. 85 Geyh, above n 8, at 725. 86 Geyh, above n 8, at 725. 87 Simon Shetreet "Judging in Society: The Changing Role of Courts" in Simon Shetreet The Role of Courts in Society (Martinus Nijhoff Publishers, Dordrecht, 1989) 467 at 467. 21 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD its dispute resolution service affects every member of society, not just the litigants that appear before the court. Consequently, the judiciary's customers should not be limited to litigants, but should encompass the jurisdiction's whole society. 3 The public: generally One's perspective of the judiciary's role may affect how one conceptualises the public whose confidence may materially impact upon the judiciary's legitimacy. The foregoing discussion demonstrated that the public can be taken to encompass each and every member of society. The ensuing question is: what fosters the public's confidence in the judiciary? B What Fosters the Public's Confidence in the Judiciary? In adopting a wide view of the public, a wide view of what fosters their confidence may also be required. Judicial impartiality has been recognised as a key driver of the public's confidence in the judiciary. Implicit in this notion is that judicial process is important because impartiality is concerned with how the law is applied to a dispute rather than what the law is. Researchers at the University of Otago conducted empirical research on public perceptions of the New Zealand court system. 88 When respondents were asked whether they believed that they "would get a fair hearing in the New Zealand court system", 89 the mean response was a 2.4 out of 5.0.90 This shows that, on average, more respondents perceived the court system as fair than unfair. From a layman's perspective, these findings may seem counterintuitive because one may assume that people who do not win a dispute would be more inclined to perceive the courts negatively, and vice versa, to average out to an even half-and-half perception of fairness. This suggests that a favourable decision may not be the only factor that determines the public's idea of fairness. To better understand what aspects of the court system promote perceptions of fairness, it is useful to compare and contrast the dispute settlement rhetoric between the judiciary and the public. 88 Saskia Righarts and Mark Henaghan "Public Perceptions of the New Zealand Court System: An Empirical Approach to Law Reform" (2010) 12(2) Otago Law Review 329. 89 Righarts and Henaghan, above n 88, at 334. 90 Righarts and Henaghan, above n 88, at 335: "a 1 represents a 'strongly agree' (a positive response to each question), a 5 represents a 'strongly disagree' (a negative response to each question), and a 3is a neutral response (neither agree or disagree)." 22 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD 1 Dispute settlement from a judge's perspective Judges are decision makers. The public is a decision recipient. Social science research has found that decision makers and decision recipients view dispute settlement differently. When researching the psychology of decision makers and decision recipients, Larry Heuer found that the former pegged their perceptions of fairness to the societal outcomes produced by a decision.91 Heuer's findings held when he combined with Diane Sivasubramaniam to investigate the psychology of judges and litigants.92 Their research reiterated that judges, akin to decision makers generally, "were more heavily influenced by outcome fairness than by procedural fairness".93 Perceiving fairness based on a societally just outcome is also termed distributive justice. Due to a lack of substantive knowledge of the law, it may be difficult for the public to properly assess whether a judge delivered a fair outcome. For example, in the judicial recusal context, judges harbour varying views as to how the fair-minded lay observer test should be applied. If judges cannot come to a consensus on whether the outcome of a recusal motion was fair, it is unrealistic to expect that the public will. Judicial preoccupation with distributive justice is understandable because judges pride themselves on applying the law accurately. However, in doing so, there is a danger that judges may not adequately consider how the public, as decision recipients, perceive the fairness of dispute settlement. 2 Dispute settlement from the public's perspective While distributive justice is undoubtedly important to the public as it may affect them directly, if not indirectly, it can be difficult to objectively assess; distributive justice to one may be distributive injustice to another. The difficulty is exacerbated when considering that recipients of the judiciary's decisions include each member of society. Significantly – and perhaps as a reflection of the difficulties in assessing distributive justice – social science research has found that people "care more about the fairness of the process that produces the outcome". 94 In other words, the public place an equal, if not greater, emphasis on procedural justice as they do on distributive justice. This theory originates from John Thibaut and Laurens Walker's seminal social science research that demonstrated people's assessments of the fairness of third-party decision-making 91 Larry Heuer "What's Just About the Criminal Justice System? A Psychological Perspective" (2005) 13 Journal of Law And Policy 209 at 214–218. 92 Diane Sivasubramaniam and Larry Heuer "Decision Makers and Decision Recipients: Understanding Disparaties in the Meaning of Fairness" (2007) 44(1) Court Review 62 at 65. 93 Sivasubramaniam and Heuer, above n 92, at 64. 94 John M Greacen "Social Science Research on 'Procedural Justice': What Are the Implications for Judges and Courts?" (2008) 47(1) The Judge's Journal 41 at 41. 23 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD procedures shape their satisfaction with an outcome. 95 Professor Tom Tyler further confirmed that "it is clear … citizen assessments of the justice of the procedures used by legal authorities to make decisions influence reactions to those decisions." 96 It also reflects the philosophical importance James White placed on procedure when stating that, "we [the public] are entitled not to 'like results' but to 'like process'."97 Four factors have been identified as the primary drivers of people's procedural fairness judgement: "opportunities for participation; the neutrality of the forum; the trustworthiness of the authorities; and the degree to which people receive treatment with dignity and respect". 98 Applying the procedural justice notion to judicial recusal, litigants and the public are likely to place an equal, if not greater, level of importance on the process by which a recusal motion is adjudicated as they are on its outcome. Therefore, if the process invites participation and appears neutral with a trustworthy judge that treats the participants with respect, litigants and the public alike are more likely to accept the outcome of a recusal motion. Increased procedural satisfaction may thus lead to increased public confidence in the judiciary, which the foregoing discussion suggests may promote its legitimacy. To assess whether judicial recusal law adequately fosters procedural satisfaction in the public, it is helpful to look for guidance from legal process theory. The following Part will set out the fundamental aspects of third-party adjudication that legitimises its outcomes. V Adjudication: A Process-based Perspective Litigation is a form of third-party dispute resolution where parties take their disputes to courts for judges to determine an outcome according to relevant law. Professor John Allison finds that "many procedural elements found in judicial and administrative adjudication perform a surrogate legitimation function." 99 Allison identified the 95 J Thibaut and L Walker Procedural Justice: A Psychological Analysis (Lawrence Erlbaum Associates, 1975). See generally: Greacen, above n 94; and Tom R Tyler "Social Justice: Outcome and Procedure" (2000) 35(2) International Journal of Psychology 117. 96 Tom R Tyler "What is Procedural Justice? Criteria Used by Citizens to Assess the Fairness of Legal Procedures" (1988) 22(1) Law & Society Review 103 at 104. 97 James Boyd White Heracles' Bow: Essays on the Rhetoric and Poetics of the Law (University of Wisconsin Press, Wisconsin, 1985) at 134. 98 Tyler, above n 96] at 121. At 121–122: participation refers to providing parties "an opportunity to make arguments about what should be done to resolve a problem or conflict"; neutrality refers to parties' beliefs "about the honest, impartiality, and objectivity of the authorities with whom they are dealing"; trustworthiness refers to parties being "concerned about the motivation underlying the decisions made by the authority with whom they are dealing"; andtreatment with dignity and respect refers to parties "having respect shown for their rights". 99 John R Allison "Ideology, Prejudgment, and Process Values" (1994) 28 New England Law Review 657 at 682. 24 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD procedural elements to generally include: "published rules, party participation, reasoned decisions, and communicated rationales". 100 He noted that adjudication processes that exhibit these features usually "have the intended and actual effect of enhancing public perceptions of legitimacy."101 Professor Amanda Frost subsequently looked to Allison and other process theorists to extract "five procedural components of adjudication that are universally considered essential to the legitimacy of the final product [the decision]".102 Frost specifically looked to the judicial process when she articulated the components as being: (1) (2) (3) (4) (5) litigants initiating disputes; the disputes being presented via an adversarial system where the competing parties articulate their conflicting views; a reasoned decision should be provided; decisions must be consistent with the rule of law; and the arbiter must be impartial. While New Zealanders do not enjoy an inalienable right to due process like Americans do, proper process is nonetheless a fundamental right protected by the Bill of Rights Act 1990.103 This Part will examine Frost's five procedural components and illustrate that they feature prominently throughout New Zealand's litigation process. A Litigants Initiating Disputes Courts do not initiate disputes in New Zealand. They rely on injured parties to bring disputes to them. The courts' passiveness stems from two limitations. First, the courts are fiscally limited to the resources and manpower that the Ministry of Justice affords them. Judges have one or two clerks and the court may have some further research and general administration assistance. This does not leave residual resources available to investigate and commence proceedings. Second, in upholding the doctrine of judicial independence, courts do not have a prerogative from anyone to instigate a dispute. Judges have no constituency from whose needs and wants an agenda can be drawn up. Nor do 100 Allison, above n 99, at 682. Allison, above n 99, at 682. 102 Amanda Frost "Keeping Up Appearances: A Process-Oriented Approach to Judicial Recusal" (2005) 53(3) Kansas Law Review 531 at 555–556. 103 There are two due process clauses in the United States Constitution. The fifth amendment to the Constitution guarantees "nor shall any person … be deprived of life, liberty, or property, without the due process of law." The fourteenth amendment to the Constitution guarantees "nor shall any State deprive any person of life, liberty, or property, without due process of law." 101 25 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD they have the expertise to isolate and address specific societal problems; they are generalists, a virtue that is more or less revered.104 It is the responsibility of the executive to bring claims on behalf of the State. It has the capabilities and the mandate to undertake this role. By requiring parties to originate disputes, it engages them in the process from the beginning and increases their feeling of participation – a contributory factor to an appearance of procedural justice factor, as Greacen identified. One commentator stated that "participation clothes the resulting decision with legitimate authority to bind the litigants". 105 It is standard practice throughout the litigation process for parties to initiate disputes, whether it be a party filing a statement of claim or applying for leave to appeal a decision, it is the plaintiff, prosecution or appellant that commences a dispute. B An Adversarial System of Presenting Disputes The traditional adversarial litigation system enables parties to present their arguments and advocate for their respective claims. Christopher Peters articulated the party-centric adversarial model when stating that "each litigant, not the court itself, locates relevant facts and identifies relevant legal authorities, and each litigant determines whether and how to present those facts and authorities to the court in the form of legal arguments." 106 Frost identified a benefit of this process by analogising that "party control over casepresentation is legitimating for much the same reasons that party control over caseinitiation is legitimating."107 By putting their case, the litigants' feeling of participation in the proceeding increases, which again contributes to an appearance of procedural justice. The decision maker is also more likely to be better informed if he or she is exposed to the competing views of the parties. Fiscal constraints also render it impractical for courts to undertake an inquisitorial or investigative role here. The judge remains "remains relatively passive"108 throughout this process and only after the parties have put their case will the judge "render a decision that is responsive to the proofs and arguments made by the litigants".109 104 The virtues of judicial specialisation is not a debate this paper seeks to partake in. Some view specialisation, particularly in certain areas such as commercial or tax litigation as a positive step. 105 Christopher J Peters "Adjudication as Representation" (1997) 97(2) Columbia Law Review 312 at 374. 106 Christopher J Peters "Persuasion: A Model of Majoritarianism as Adjudication" (2001) 96(1) Northwestern Law Review 1 at 20. 107 Frost, above n 102, at 559. 108 Peters, above n 106, at 21. 109 Peters, above n 106, at 21 (emphasis added). 26 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD C Reasoned Decision-making Frost recognised that decisions accompanied by logical and coherent reasoning are "a vital source of legitimacy for judicial decision-making". A number of justifications for the reasoned decision imperative will be discussed. First, as noted, the judiciary's legitimacy does not originate from a democratic mandate. Rather, it is the public's confidence in its function that legitimises the judiciary. A reasoned decision illustrates that the judge has factored in the parties' arguments, which bolsters the appearance of neutrality for litigants to help further their perception of procedural justice. It goes some way in appeasing any concerns a losing party may have about the judge holding prejudicial or arbitrary views that could have materially affected the outcome of the case. Second, Professor Frederick Schauer clearly expresses the cognitive value in providing reasons for a decision. He stated that a legally, socially or morally impermissible result may arise from legally, socially or morally impermissible reasoning. 110 Schauer opines that "perhaps there are things we [including judges] can think but cannot write down."111 If so, one may be able to reason impermissibly if thought alone was one's only constraint. But, when one has to commit to that thought by way of orating it or recording it in prose, a further constraint is added; a constraint that limits one's ability to reason impermissibly. It commits a decision maker to the reasoning that he or she articulates, which is more likely to lead to permissible reasoning due to the possibility of subsequent scrutiny. Accordingly, requiring a judge to provide reasons for a decision imposes a constraint on judicial discretion to adjudicate within a permissible framework that may or may not otherwise be there. Finally, Schauer raises the interesting proposition that "when we [including judges] provide a reason for a particular decision, we typically provide a rule, principle, standard, norm, or maxim broader than the decision itself". 112 The decision that the reason produces is just one of a number of factual matrices that could be decided under that enunciated rule. Thus, in conjunction with the said idea of positively constraining thought, reasoned decisions are more likely provide consistency in decision-making. Reasoned decisions are also necessary to enable a party to exercise their right of appeal. Without reasons, it is impractical – and realistically untenable – for a litigant to base an appeal on the simple notion that the trail judge was "wrong". Chief Justice Elias also 110 Frederick Schauer "Giving Reasons" (1995) 47 Stanford Law Review 633 at 652. Schauer, above n 110, at 652. 112 Schauer, above n 110, at 641. 111 27 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD highlights the importance of access to decisions in vindicating the judiciary's conduct. 113 In New Zealand, reasoned judgments are delivered and publicly available for all High Court, Court of Appeal and Supreme Court cases. D Recourse to the Law Having recourse to the law in adjudicating a dispute is an uncontroversial constraint on judicial power.114 In leading the legal process movement, HLA Hart recognised that "rule by arbitrary choice is not rule by law". 115 With the law becoming more complex and indeterminate, judicial discretion plays an increasingly material role in decisions. 116 The neutrality and trustworthiness of a judge is likely to be questioned if he or she exercises this discretion ad hoc without rooting it in the law. The strong emphasis that the common law places on precedent further constrains the judge to resolve like disputes with like decisions. Parties' perceptions of neutrality and trustworthiness will also be enhanced if they are confident that a judge has decided a dispute according to the law. Outcomes adhering to the rule of law are more likely to provide rational, consistent and predictable decisions. This enables the law to develop logically and in a manner that reflects the changing values of society. Predictability helps guide societal behaviour by providing a distinction between what is legal and what is illegal. Consistent decisions are likely to improve litigants' perceptions of due process because if similar facts produce similar decisions, a litigant would perceive the process by which the decisions was made. E An Impartial Arbiter Arguably the most fundamental facet of any adjudication and litigation process is that disputes are resolved by an impartial arbiter. The whole premise for judicial recusal is founded on an impartiality imperative; if partial judges were accepted, there would be no need for judges to consider not sitting on a case due to a reasonable apprehensions of bias. As Tipping J enunciated in Saxmere No 2, "the rationale for the rule against apparent bias is to ensure that public confidence in the legal system and the impartiality of judges is 113 Elias, above n 10, at 5–6. Joseph, above n 74, at 153–199. 115 Geoffrey C Shaw "HLA Hart's Lost Essay: Discretion and the Legal Process School" (2013) 127(2) Harvard Law Review 666 at 668. 116 This notion goes against the traditional legal formalist theory of there always being one answer to a question and so discretion or morals should not play a part in decision-making; decision making is thus purely based on competency and if judges are competent, they should all come to the same outcome on any given dispute. As the law has developed since this articulation, it is now widely regarded as overly simplistic and unrealistic. 114 28 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD maintained."117 Thus, if litigants – and the public – harbour a reasonable apprehension of bias of a judge, a proceeding's neutrality may be compromised, as well as the parties' trustworthiness of the judge. The effects may also flow on to parties limiting their cooperation and participation in the proceedings and thereby affecting perceptions of procedural justice. An overview of the cognitive difficulties with self-adjudication is useful to illustrate the psychological premise for impartial adjudication. Common sense suggests that "[a] biased mind rarely realises its own imperfection".118 Ironically, the more biased a person may be, the more difficult it is likely to be for them to accurately assess their own biased. Cognitive theory provides a firmer basis for highlighting the inadequacies of self-adjudicating conflicts of interest. Researchers at Stanford University revealed that people, including judges, have a "bias blind spot".119 The blind spot refers to an asymmetry in judging one's cognitive susceptibilities vis-à-vis other people's cognitive susceptibilities. 120 This asymmetry leads to an overoptimistic view of one's competency due to one being unaware of unconscious factors that may affect one's decisions. 121 In Stanford's research, individuals reported that they were "less susceptible than their peers to various cognitive and motivational biases".122 Some may argue that the years of training and experience that judges have in resolving disputes may enable them to exercise superior cognitive abilities of adjudication than the general population. However, regardless of any superior cognitive capability, it is likely judges are susceptible to the same natural biases that all humans may be susceptible to. These biases may range from status quo bias 123 to overconfidence124 and false consensus bias.125 Preserving judicial impartiality is prevalent throughout the 117 Saxmere No 2, above n 15, at [38]. Note "Disqualification of a Judge on the Ground of Bias" (1927) 41(1) Harvard Law Review 78. 119 Emily Pronin, Daniel Lin and Lee Ross "The Bias Blind Spot: Perceptions of Bias in Self Versus Others" (2002) 28 Personality and Social Psychology Bulletin 369. 120 Pronin, Lin and Ross, above n 119, at 374. 121 Pronin, Lin and Ross, above n 119, at 374. 122 Pronin, Lin and Ross, above n 119, at 374. 123 Jeffrey W Stempel "In Praise of Procedurally Centered Judicial Disqualification – and a Stronger Conception of the Appearance Standard: Better Acknowledging and Adjusting to Cognitive Bias, Spoliation, and Perceptual Realities" (2011) 30(4) The Review of Litigation 733 at 741, n 24: status quo bias is an unconscious tendency to interpret something in a manner that maintains the existing situation. In the recusal context, it could mean that more traditionalistic judges may uphold the strong presumption of impartiality by overemphasizing their duty to sit. 124 Stempel, above n 123, at 741, n 25: overconfidence, as alluded to earlier, is when people, including judges, tend toward excessive optimism and overconfidence. In the recusal context, this may lead to a judge overemphasising their ability to detach themselves from any underlying interests. 125 Stempel, above n 123, at 741, n 26: false consensus bias arises when a person assumes that everyone else interprets or perceives something the same way as they do, even if it is ambiguous. In the recusal context, a judge may feel that an interest need not be disclosed to the parties because both parties would simply view it as immaterial, as he or she does. 118 29 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD litigation process with parties being able to file recusal motions at any stage of the proceeding, subject to implied waiver, and appeal to have a judgment set aside based on impartiality. F Process-based Adjudication: Generally Although these five key aspects of judicial procedure have been described independently, the majority are interdependent. For example, it will be more difficult for a judge to be partial if a decision must be accompanied by reasoning as the parties and public can scrutinise the reasoning. Moreover, if that decision is subject to precedent, there is even less scope for extra-legal influences to affect an outcome because the case for departing from precedent must be strong. Conversely, it will be difficult to be assured that the judge had sufficient recourse to law if no reasons are provided for their decision. Likewise, if the dispute, the facts and the arguments are solely brought by the parties, it is less likely that a judge's personal agenda or motives can shape the outcome because the dispute must be addressed in the manner that the litigants have put it. These processes are commonly followed throughout civil litigation in New Zealand and moreover, they are followed throughout the majority of third-party adjudicatory processes. There may be instances, such as interim injunctions, where due process may be marginalised due to temporal urgency, but these instances are few and far between.126 Departing from established procedural methods is the exception, not the rule. Individually and collectively, these procedural elements help to legitimise the judiciary's adjudicative role which in turn nurtures the public's confidence in the third branch of government. In light of this, the following Part illustrates the extent to which judicial recusal process strays from these fundamental qualities. VI Judicial Recusal: A Mismatch between Theory and Practice With the soft and indeterminate nature of existing recusal procedures outlined and five key practices that legitimise adjudication analysed, it is possible to compare and contrast the former with the latter to ascertain whether current recusal procedure follows best practice. This Part will use legal and psychological rhetoric to illustrate the "procedural vacuum" 127 in which judicial recusal operates. 126 In the case of interim injunctions, any marginalisation of proper process is subsequently rectified following a full trial. 127 Frost, above n 102, at 551. 30 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD A Impediments to Litigants Initiating Recusal Motions As a result of information asymmetries, the onus is on a judge, rather than a litigant, to raise any potential conflicts of interest.128 Prima facie, this does not reconcile well with best practice adjudication because a judge should not initiate a dispute. However, in disclosing an interest, a judge is not necessarily initiating a dispute. Rather, they are merely providing the information of an interest that then may form the basis of a recusal motion; it is still the litigant who raises and disputes an issue with a disclosed interest. Further, the usual concerns of fiscal deficiencies and a lack of mandate are absent in burdening a judge with disclosure; the judge inherently has the knowledge of the interest and need not conduct further research into the interest. Unlike issuing proceedings, amending statements of claim or submitting interlocutory appeals, there is little guidance as to how a recusal motion can or should be made. The usual method of giving notice is through the Registrar or by filing an originating application. A lack of formality may reasonably conjure some hesitancy in counsel to bring a recusal motion for fear of offending a judge. Richard Flamm analogised questions of legal error with questions of partiality when he observed that "[just] as judges generally do not like to admit having committed legal error, they are typically less than eager to acknowledge the existence of situations that may raise questions about their impartiality." 129 An understandably conservative approach to filing recusal motions may to ensue. Litigants may be faced with a compromise between fully participating in a proceeding by filing a recusal motion and a consequent lack of judicial neutrality if the motion is dismissed and the judge is annoyed. This is undesirable because litigants should not dissuade themselves from filing recusal motions for fear of subsequent adverse findings by a challenged judge. B A Lack of Adversarial Procedure Conflicts of interest usually arise between a judge and one party. Thus, to align with the adversarial process, a judge should offer their conflicting views when putting their case to the party that has made a recusal motion as to why recusal is not required. However, a judge rarely responds to a recusal motion as a defendant or respondent would respond to a statement of claim due to the judge also being the arbiter of the motion. It goes against common sense and cognitive theory to expect a judge to be able to argue for a particular outcome in a dispute and then be able to impartially adjudicate that very dispute. Without the adversarial forum for all parties to articulate their views, there is a 128 "Guidelines for Judicial Conduct", above n 44, at [41]–[45]. Richard E Flamm Judicial Disqualification: Recusal and disqualification of Judges (2nd ed, Banks & Jordan Law Publishing Co, Berkley, 2007) at 25. 129 31 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD danger that a recusal motion could be decided without the arbiter being exposed to the parties' competing arguments and thus not being fully informed. This may endanger litigants' trust that the judge is doing what is right for them as well as marginalise their participation by not being able to respond to arguments as they would with other parties. C A Dearth of Reasoned Decisions and Precedent The majority of recusal decisions provide little insight into the nature of the interest in question, the grounds on which recusal was sought or the judge's rationale in deciding to sit or not to sit. This is especially so when a judge recuses themselves sua sponte before proceedings commence or even before they are listed on a case. Although prophylactic measures to avoid wasted litigation time is preferable, a judge's decision should be subject to scrutiny, as other judicial decisions are. The absence of a decision precludes public scrutiny and cognitive commitment, which may also invite a judge to consider impermissible factors. Professor Jeffrey Stempel recognised the shortage of reasoning when he stated that "the clipped, abrupt, and uninformative manner in which many disqualification decisions are delivered undermines the public confidence the process should inspire". 130 Precedent plays an important role in guiding judges to an accurate judgment. It is particularly useful when the law is ambiguous, like the fair-minded lay observer test. The upshot of judges providing little to no reasoning for their recusal decisions is that precedents are scarce. Adding to the inadequacy is the inclination for judges to provide more reasons for dismissing a recusal motion than for affirming a recusal motion. 131 Without reference to precedent, people may more readily perceive a judge to be adjudicating a recusal motion ad hoc rather than by the rule of law. D The Archetypical Interested Arbiter A challenged judge self-adjudicating recusal motions is arguably the most glaring deficiency of the current judicial recusal process. The process is often justified by the idea that a challenged judge possesses the most knowledge about the interest in question and thus they are in the best position to assess whether it will appear to affect their impartiality. 132 This is a flawed proposition that wrongly correlates knowledge with adjudicative ability. 133 Cognitive theory reinforces the challenges associated with 130 Stempel, above n 123, at 799. Frost, above n 102, at 570. 132 Hammond, above n 25, at 83. 133 Geyh, above n 8, at 708. Geyh articulated the fundamental fallacy of judicial recusal law by stating that: "disqualification practice proceeds on two implicit assumptions: that judges are able to assess the extent of 131 32 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD expecting judges to be able to impartially adjudicate questions of partiality made against them. Throughout the litigation process, a disinterested judge with less intimate factual knowledge than the parties adjudicates the case. While this bolsters the public's perceptions of neutrality, it is difficult to envisage that self-adjudication of recusal motions has the same effect. When commenting on this practice, Hammond concluded that "there is a hopeless tension between the state-fostered guarantee of a neutral and objective arbiter of a case and the state's current process arrangements for disqualification decisions."134 E The Shortcomings of Current Judicial Recusal Procedure Notwithstanding that litigants may be discouraged from questioning a judge's partiality, if a recusal motion is made, an absence of adversarial procedure, reasoned decisionmaking, precedent and an independent arbiter highlights the discrepancies between best practice found in civil litigation and the deficiencies of judicial recusal procedure. The fundamental importance of judicial impartiality in fostering the public's confidence in the judiciary justifies a look at taking a more principled approach to addressing questions of judicial partiality. VII A Way Forward: Procedural Reform In keeping with this paper's focus on process over substance, the author proposes procedural reform to provide more clarity, certainty and custom for judicial recusal. The following reform is to be imposed by the judiciary, rather than through legislative intervention. As Hammond emphasised, maintaining a separation of powers and judicial independence is vital, particularly in an area like impartiality which goes to the heart of the judiciary's credibility. 135 To justify a formal regulatory regime, Hammond argued that the judiciary must be "beyond self-redemption". 136 The author concurs with Hammond in observing that the New Zealand judiciary is not nearing that point. Rather, it has long enjoyed the public's confidence. Thus, reform should come from within the bench, not from the outside. their own bias; and that judges are able to assess how others reasonably perceive their conduct. Neither assumption is safe." 134 Hammond, above n 25, at 148. 135 Hammond, above n 25, at 153. 136 Hammond, above n 25, at 153. 33 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD The adopted processes should be recorded in the Gazette so that the legal profession and the public can be fully aware that the judiciary acknowledges and is actively taking steps to maintain judicial impartiality and preserve public confidence. A Disclosure and Filing Recusal Motions 1 Disclosure McCoy observed that "the vulnerability of the parties before the court is their dependence on the timeliness, comprehensiveness and reliability of judicial disclosure."137 While the New Zealand judiciary's Guidelines for Judicial Conduct provide some useful advice to judges of the importance of disclosure and factors to consider when disclosing an interest, the guidance is somewhat abstract.138 A register – similar to that which members of the executive and legislature are subject to – whereby judges would be required to periodically disclose their pecuniary interests to the public has been suggested as a means of improving disclosure and transparency. 139 This paper does not view public financial disclosure by judges as the way to symmetrise the asymmetry of information between the bench, the bar and the public. Instead, this paper looks to impose greater disclosure obligations on judges to require them to disclose any pecuniary, personal or professional relationships with parties in open court. This does not require a significant change from the status quo, rather it is a hardening and nuancing of the existing duty to disclose. Currently, judges may withhold information unless, after introspection of the nature of the interest and its proximity to the parties, they perceive the interest to be potentially conflicting. Essentially, this is a negative duty that begins from the basis that a judge is impartial – and thus nothing need be disclosed. The duty only requires a departure from this position if a judge, in unilaterally undertaking a relevance test of any material interests, considers it necessary. Clearly, a judge's introspection determines whether an interest is disclosed. The foregoing psychological analysis portrayed the cognitive limitations associated with self-assessment, which may lead to an overly conservative approach to disclosure. Judges may unduly perceive themselves to able to detach personal or professional interests from a dispute and adjudicate with an impartial mind. In doing so, judges may also attribute their perceived abilities to the public to conclude that disclosure is unnecessary. Moreover, John Leubsdorf found that the 137 McCoy, above n 14, at 335 (emphasis added). "Guidelines for Judicial Conduct", above n 44, at [41]–[45]. 139 Register of Pecuniary Interests Bill 2010 (240–1). See generally: Justice and Electoral Committee Register of Pecuniary Interests of Judges Bill (21 February 2014); Law Commission Review of The Judicature Act 1908: Towards a New Courts Act (NZLC R126, 2012); Law Commission Towards a New Courts Act – A register of judges' pecuniary interests? (NZLC IP21, 2011). 138 34 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD "appearance [of bias] test invites judges to rest on appearance, instead of looking deeper [during introspection]".140 Leubsdorf identified that inadequate disclosure is, in part, due to "systematic inaccuracy caused by unconscious leanings not removable by introspection". 141 To minimise any such limitations and encourage disclosure, the author hypothesises that the existing negative disclosure duty should be framed more positively with a softer starting position. Currently, the strong emphasis on impartiality as the base point from which disclosure is made may create some disclosure inertia in judges. If a more party-oriented starting point is adopted, less emphasis may be placed on judicial introspection. For example, if a judge readily discloses any potentially conflicting interests before unilaterally undertaking a materially or relevance test of the interests, the possibility of systematic inaccuracy in introspection may be reduced. It is envisaged that this approach will widen the ambit of disclosure and the onus that was previously on a judge's introspection will partly be shifted to the parties, which should limit the scope for misjudgement. Although wider disclosure may delay proceedings, the time that may be saved from minimising the possibility of a material interest being subsequently discovered and the ensuing potential for wasted litigation is significant. Moreover, with increased disclosure and transparency, litigants' trust in judges and their neutrality may be bolstered. 2 Process of questioning an interest and filing a recusal motion A formal and uncomplicated procedure for litigants to file recusal motions should be instilled. Simply raising concerns via notice to the court's Registrar or via an originating application does little to explicitly recognise litigants' right to question a judge's impartiality. A specific avenue to file a recusal motion, similar to that of an interlocutory appeal, should be sufficient and not overly burdensome to remedy the lack of clarity. Recognising questions of partiality as a standard part of court proceedings may go some way to normalising the practice. As a result, the trepidation litigants may harbour of provoking a judge by questioning an appearance of partiality and the corollary stigma that judges may feel when their impartiality is questioned is likely to be reduced. Further, a recusal motion should have to be brought as soon as practically possible after a relevant interest is disclosed. The complexity of the interest or the subsequent questioning of the judge that a party may have to undertake will affect the practicality of how soon a recusal motion can be brought. The existing implied waiver protocol 140 John Leubsdorf "Theories of Judging and Judge Disqualification" 62(2) New York University Law Review 237 at 278. 141 Leubsdorf, above n 140, at 262. 35 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD adequately covers this. If material detail regarding a judge's potentially conflicting interest subsequently arises, the implied waiver will not apply and parties will be entitled to file a recusal motion despite the delay. This is intended to not only minimise the possibility of wasted litigation, but also to promote proper disclosure of interests as partial disclosure may perpetuate a reasonable apprehension of bias if the judge is later found to be withholding material information. B An Impartial Arbiter The fallacy of a challenged judge being in the best position to assess a conflict of interest is clear.142 The New Zealand judiciary has attempted to lessen this untenable position by suggesting that "in cases of uncertainty it may be desirable for the judge to discuss the matter with the relevant head of jurisdiction or another judge." 143 Likewise the Court of Appeal Recusal Guidelines state "where the issue is not clear cut, the judge should consult … with other members on the panel". 144 However, the author opines that consultation with other judges is insufficient for three reasons. First, it can be difficult for judges that are not well versed in the factual matrix of a case to be able to provide reliable counsel ad hoc, particularly when they are unlikely to have the benefit of the motioning party's views to reconcile the challenged judge's views. 145 Second, consultation may be impractical in trial courts where a single judge sits on a case and other judges on the court may be preoccupied with cases they are sitting on. Third, any such consultation is confidential. Parties may have no knowledge that a challenged judge has consulted with their colleagues, let alone the content of any consultations. Thus, confidentiality is unlikely to further the appearance of justice. To resolve this preeminent procedural shortcoming, recusal motions need to transferred to and decided by an independent judge.146 142 See Part V(E). "Guidelines for Judicial Conduct", above n 44, at [29]. 144 "Court of Appeal Recusal Guidelines", above n 56, at [2(b)]. 145 If a challenged judge wishes to consult a potentially disqualifying interest with another judge in a trial court, the judge being consulted will not be informed of the facts of the interest or the case. The situation is different in an appellate court where a panel of judges will be sitting. Here, a challenged judge may be able to consult with the other panel members in a more informed manner as they will presumably be versed in the facts of the case. 146 When a recusal motion is brought against a judge, their credibility is at stake. Accede to the motion too readily, for example when a fair-minded lay observer would not reasonably apprehend any apparent bias, and litigants may think the judge is "soft" or inept at detaching personal interests. Conversely, if a challenged judge rejects the recusal motion, they are vulnerable to criticism of their impartiality and of course, to a subsequent appeal. 143 36 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD This paper proposes that once a litigant files a recusal motion, an independent judge should be called in to hear the motion and conduct the necessary proceedings.147 In a trial court, such as the High Court, a motion should be transferred to a disinterested colleague from the same registry as the challenged judge. A robust method of selecting who that independent judge is will need to set up. One possibility is to form a standing recusal committee in each registry consisting of two or three judges, depending on the size of the registry, that rotate every six to twelve months. If it is impractical for a judge on a standing committee to hear a recusal motion, the next judge due to sit on the committee should be summoned. Of course, the independent judge to whom a motion is transferred can also recuse themselves sua sponte if they cannot, or appear to not be able to, bring an impartial mind to adjudicate the motion. In an appellate court, such as the Court of Appeal, where there is a panel of judges, the other members of the panel should hear the motion. The challenged judge should not sit on the panel whilst the other panel members hear the motion. Likewise, in a court of last instance, such as the Supreme Court, where the court sits as a whole, the remaining members of the court should hear the motion without the challenged judge sitting on the panel. In the event of a split decision, the head of the relevant court should rule on the matter. This will not undermine the validity of proceedings because courts of final instance can often hear procedural matters without a full quorum. 148 To dampen litigants' fears about a judge taking offence to being challenged, the party submitting the recusal motion should be anonymous.149 To ensure anonymity, all submissions should be made to the Registrar in writing, who will then pass them on to the disinterested judge. Oral hearings would reveal the complainant to the judge and so the proceeding should wholly occur in writing. Under this regime, judges will still be able to recuse themselves sua sponte. Although this may seem counterintuitive to the idea of having an independent adjudicator, it should not compromise the process. If a judge perceives that an interest may raise a reasonable apprehension of bias, it is likely that an independent judge would come to a similar conclusion. Enabling the challenged judge to conduct this prima facie assessment saves court resources and limits delays in proceedings. The prescriptive nature of the proposed transfer regime reflects the desire for harder rules that provide the bench and litigants with more certainty and guidance. It also 147 Proceedings in a recusal motion could include affidavits from the applicant and the judge, as well as an articulation of both parties' views. 148 Supreme Court Act, s 28. 149 See Part VI(A) for discussion regarding the fear litigants may face in questioning a judge's impartiality due to the potential for subsequent retribution by the judge. 37 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD helps to bring judicial recusal in line with other civil procedures that involve formal processes. C Positive Externalities of an Independent Judge: Adversary; Reasoned Decisions; and Precedent Transferring a recusal motion to an independent judge enables an adversarial process, reasoned decisions and precedent to develop. 1 Adversarial process Without having to adjudicate a recusal motion, a challenged judge is in a more practical position to offer their arguments for continuing to sit on a case. This should lead to more informed considerations of whether an apprehension of bias arises because a challenged judge can contextualise the interest in a manner that is currently missing due to an absence of adversarial process. 2 Reasoned decisions generating precedent An independent judge adjudicating a recusal motion is well equipped to reconcile the arguments of a challenged judge and a complainant according to law. In doing so, the judge should articulate their reasoning. Judges who recuse themselves sua sponte should also be required to provide reasons for their decision to not sit on a case. This is particularly important in light of the lopsided recusal precedent that is currently available. Developing a comprehensive body of precedent will not only enhance the uniformity of recusal adjudication but also provide guidance and predictability to litigants with regards to the types of interests that may justify recusal. VIII Procedural Reform: A Critique In contemplating the proposed reform, there were a number of options that were foregone to come to the regime that has been set out. The following section will address potential drawbacks of the proposed reform as well as exploring some alternatives that were considered, but not adopted. A Concerns with Transferring Recusal Motions to an Independent Judge 1 Practical concerns Increased pressure on already stretched judicial resources is the practical concern with transferring recusal motions to independent judges. Unlike a challenged judge, an 38 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD independent judge will require time to become accustomed with the interest in issue. The proposed disclosure regime may go some way to minimising the time an independent judge requires to fully discover the facts as all relevant interests should be disclosed at first instance. While an independent judge considers a recusal motion, the cases he or she is sitting on at the time will need to be adjourned. Thus, holding all else equal, the average length of all cases, whether they involve a recusal motion or not, is prima facie likely to increase. While undoubtedly a real concern, procedural delays can be easy to overstate, particularly when the counterfactual is not considered. A transfer to an independent judge will only occur if a recusal motion is filed; there will be no increased delay relative to the status quo if a judge recuses themselves sua sponte. Further, a judge’s ability to synthesise fact and law to come to an efficient solution should not be underestimated; judges are well trained to undertake this task – it is their job. An argument could also be made that parties may be less inclined to appeal the dismissal of a recusal motion made by an independent judge when compared to a dismissal made by a challenged judge. 150 It is difficult to quantify the increased resource requirements and procedural delays of transferring recusal motions to independent judges. No common law jurisdictions employ this procedure. Although it may be inaccurate to attempt to draw meaningful conclusions regarding an undue increase in demand for court resources ex ante, one commentator, when contemplating a similar regime to what has been proposed here, suggested that, on balance, "the overall administrative burden in fact might not increase". 151 2 Principled concerns Cognitive studies have established that judges, like everyone else, are susceptible to unconscious biases that hinder their ability to self-adjudicate.152 Bearing in mind that an independent judge to whom a recusal motion will be transferred is a colleague of the challenged judge, is he or she going to be any more effective in applying the fair-minded lay observer test than the challenged judge? Akin to a litigant's hesitancy when making a recusal motion, an independent judge may also be cautious in affirming a recusal motion for fear of offending their colleague.153 Additionally, Geyh theorised that transferring a 150 Seth E Bloom "Judicial Bias and Financial Interest as Grounds for Disqualification of Federal Judges" (1985) 35 Case Western Reserve Law Review 662 at 697. 151 Bloom, above n 150, at 697. 152 See Part V(E) for discussion on the psychology of self-adjudication. 153 While this finding should not be taken at face value due to contrast in legal systems between New Zealand and the United States, empirical research from the United States provides an interesting insight into judges' psychology of assessing their colleagues' impartiality. Jeffrey M Shaman and Jona Goldschmidt Judicial Disqualification: An Empirical Study of Judicial Practices and Attitudes (American Judicature Society, Nashville, 1995) at 70: "under the same circumstances judges … [have a] stronger tendency to disqualify themselves than to recommend disqualification of a colleague". It is not 39 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD recusal motion to an independent judge may have a detrimental effect on a challenged judge’s credibility because "assigning disqualification motions to a different judge implies that the target judge cannot be trusted to rule impartially". 154 In acknowledging that these are cogent concerns about the (in)efficacy of an independent judge adjudicating a recusal motion, it is pertinent to return to first principles to fully address their validity. Impartiality is assessed from the perspective of a fairminded lay observer. It reflects Lord Hewart CJ’s well-cited dictum of justice being seen to be done. Thus, notwithstanding the merits, or lack thereof, of an independent judge's decision, the public's perception of how that decision was made is of utmost importance. Accordingly, the public is more likely to think that a decision is made impartially if it is made by an independent judge as opposed to being self-adjudicated by the challenged judge. Furthermore, judges are accustomed to disagreeing with one another and having colleagues critique their judgments. For example, it is commonplace for judges to dissent from the majority in appellate courts. Similarly, it is standard practice for an appellate court to overturn a trial court's decision. Having a decision overturned is arguably more critical of a judge than a finding of apparent bias because the former speaks to a judge's competency and aptitude to adjudicate. While genuine questions remain about the actual benefits of transferring a recusal motion to an independent judge, the perceived benefits should be sufficient to justify transferring recusal motions to independent judges. B Alterations to the Proposed Reform 1 When should a recusal motion be transferred to an independent judge? The proposed reform suggests that all recusal motions should be heard by an independent judge. Contrarily, Hammond envisages that, at first instance, all recusal motions should be determined by the challenged judge;155 only if a recusal motion is dismissed should it be transferred to an independent judge or, in an appellate court, to the other members of the sitting panel. 156 Interestingly, in the latter situation, Hammond saw no difficulties in a challenged judge continuing to sit on a panel and hear the recusal motion even after dismissing it at first instance. 157 Presumably, Hammond was addressing resource inconceivable that New Zealand judges may think in a similar vein, especially considering the small size of New Zealand’s jurisdiction, judges are more likely to be well acquainted with each other than they are in the United States. 154 Geyh, above n 8, at 728. 155 Hammond, above n 25, at 165, appendix E. 156 Hammond, above n 25, at 148–149. 157 Hammond, above n 25, at 148–149. 40 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD constraints in suggesting this method of transferring recusal motions. While this is likely to cause less delays and require less resources than transferring all recusal motions to independent judges at first instance, principled concerns, similar to those addressed earlier, arise.158 By maintaining self-adjudication at first instance, the shortcomings of the current recusal regime will continue to prevail.159 Further, a challenged judge may feel that their credibility is harmed if their decision to dismiss a recusal motion is subsequently overturned by an independent judge – a situation that would be avoided if the recusal motion is transferred to an independent judge at first instance. Most importantly, the cognitive limitations of self-judging and the accompanying marginalisation of public confidence will continue to undermine the judicial recusal process. Accordingly, the resources that would be saved by adopting this quasi-independent recusal regime need to be balanced with the principled compromises that it entails. 2 Procedural posturing While the heightened disclosure obligation, standardised procedure for litigants to file recusal motions, increased scrutiny on that motion by an independent judge and a reasoned decision has its benefits, it may be subject to abuse. Primarily, litigants may file frivolous motions simply to elongate the proceeding and squeeze the other party's financial resources. To disincentive procedural abuse for private benefit, the proposed reform has some safeguards. To minimise delay, an implied waiver will continue to apply to prevent recusal motions being made in an untimely manner. Moreover, if an independent judge deems a recusal motion to be wholly aimed at delaying the proceeding rather than a genuine question of a judge's potentially disqualifying interest, costs for the time the recusal motion took to determine may be awarded for the party that did file the motion to ensure that they are not financially harmed by a litigant's procedural tactics. 3 Peremptory recusal To avoid potential confusion, uncertainty and inconsistency, some commentators have gone beyond procedural reform to help assess recusal motions to a system of peremptory recusal. 160 Under a peremptory recusal regime, upon an allegation of partiality being made, the case is automatically transferred to a new judge without the need to find apparent bias.161 Each party is limited to the opportunity to make one claim. 162 158 See Part VIII(A)(2). See Part VI. 160 Frost, above n 102, at 587, n 262. 161 Frost, above n 102, at 587. 162 Frost, above n 102, at 587. 159 41 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD Proponents of a peremptory system cite its simplicity, expediency and the benefit in not requiring an application of the indeterminate apparent bias test. It is based upon the idea that impartiality does not lie on a spectrum; one is either impartial or partial. Thus, when there is even an allegation of partiality, the public may question the judge's impartiality and, since impartiality revolves around public perception, the damage to the public's perceptions of impartiality has been done. A peremptory recusal regime is undeniably simpler than applying the apparent bias test to a judge's potentially conflicting interest. However, it also has major shortcomings. A peremptory recusal regime can lead to a case being reallocated twice to two new judges. While any ensuing delays need to be considered against any delays that arise from transferring a recusal motion to an independent judge, there is potential for the delays to be substantial. Further, without an articulation of the interest in question, no guidance is provided to ensure that the new judge to whom the case may be transferred will be any less partial than the recused judge. By automating judicial recusal, the number of recusals is likely to increase, which may harm public confidence in the judiciary. Frost recognised the dangers of too much judicial recusal when she stated that "increased frequency of disqualification … might arguably tend to undermine public confidence in the judiciary by disparaging the general impartiality of judges." 163 Thus, this paper opines that the peremptory recusal regime's practical and principled flaws outweigh its simplicity and expediency. C Focus on Substance Rather than Procedure This paper has looked to procedure as a means of fostering judicial impartiality and in turn maintaining the public's confidence in the judiciary. Faced with the reality that reasonable minds can differ in determining reasonable apprehensions of bias, principled procedural safeguards, rather than supplementary substantive articulation, have been used in an attempt to curb inconsistencies and increase transparency and uniformity. 164 Professor Stempel, while accepting that there is a need for greater procedural 163 Frost, above n 102, at 747. See for example: Philip Bryden and Jula Hughes "The Tip of the Iceberg: A Survey of the Philosophy and Practice of Canadian Provincial and Territorial Judges Concerning Judicial Disqualification" 48 Alberta Law Review 569. Although this is a Canadian survey, the Canadian jurisdiction similarly looks to a reasonable apprehension of bias in deciding recusal motions. The authors proposed hypothetical scenarios to which the surveyed judges responded by stating whether they would recuse themselves or not or whether they were unsure. In over half of the scenarios, no one answer attracted the support of a majority of respondents. 164 42 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD mechanisms, suggests that "the legal system and the body politic needs to accept an updated, 'post-modern,' approach to operationalising the appearance standard". 165 Stempel's proposition was based on the contradiction between the theoretical base for the appearance standard requiring "almost universal [public] agreement that the appearance of impartiality standard has been breached" 166 and the reality of a "modernpost-modern world of diverse communities, differing ideologies, varied background, and competing ideologies"167 that prevents universal consensus. He opined that recusal law is unduly conservative – and in fact weak – if broad agreement is necessary before a finding of partiality is made. 168 To remedy this, Stempel set out a need to "expand [the appearance standard] to match the reality of illusive consensus". 169 The importance of substance did not allude Geyh either, as he posited the need for the legal profession and the public to "share a basic understanding of what constitutes an appearance of impartiality". 170 This paper acknowledges the merits of Stempel's argument. In the interests of space, the focus of this paper has been on procedural reform because the author hypothesises that it is the area that is in most need of attention. This sentiment is shared by other interested parties. For example, when investigating the way forward for judicial recusal, the Law Commission identified that "there is widespread agreement amongst commentators that the least satisfactory aspect of current recusal law lies in the processes adopted, or more accurately, not expressly formulated by courts."171 The Law Commission went further in concluding that they consider "the best way to deal with potential judicial conflicts of interest is to have clear, robust and well-publicised rules and process for recusal."172 Moreover, principled procedure can aid substantive clarity. Reasoned decisions build a wealth of precedent that may provide the legal profession, and eventually the public, with the necessary clarity to harmonise their understanding of partiality. As Frost deduced, "altering the substance of the recusal standards has proven to be an ineffective method of reforming [judicial recusal law]".173 165 Stempel, above n 123, at 808. Stempel, above n 123, at 808. 167 Stempel, above n 123, at 810. 168 Stempel, above n 123, at 810. 169 Stempel, above n 123, at 811. See at 811–823 for Stempel's take on operationalising a minority recusal standard. 170 Geyh, above n 8, at 676. 171 Law Commission Towards a New Courts Act – A register of judges' pecuniary interests?, above n 139, at [2.23]; Hammond, above n 25, at 72; New Zealand Law Society "Submission to the Justice and Electoral Committee on the Register of Pecuniary for Judges Bill 2010" at 13: "current practices are not well understood by litigants and counsel, and they are inconsistently applied". 172 Law Commission Review of The Judicature Act 1908: Towards a New Courts Act, above n 139, at [6.58]. 173 Frost, above n 102, at 534. 166 43 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD D Reform: A Solution in Search of a Problem? Following the resignation of Wilson J, Dr Kennedy Graham MP introduced the Register of Pecuniary Interests of Judges Bill to parliament.174 As alluded to earlier, the Bill requires judges to periodically disclose their pecuniary interests in a public register.175 Although it looked to reform by way of disclosure rather than procedure or altering the substantive test for bias, the legal profession vehemently opposed it. The Chief Justice (on behalf of the New Zealand judiciary), the New Zealand Law Society and the New Zealand Bar Association all stated that a pecuniary interests register is unwarranted and unnecessary. 176 They, along with the Law Commission and the Justice and Electoral Committee, cited there was "no evidence of a lack of public confidence in the judiciary"177 to justify reform. This paper does not refute that the New Zealand judiciary enjoys a high level of public confidence. However, the author suggests that it can be misleading to assume that the end – with the end being public confidence in an impartial judiciary – justifies the means – with the means being judicial recusal law. It is worth noting a speech delivered by New Zealand's former Chief Justice, Sir Thomas Eichelbaum, where he stated that the judiciary "[is not] a bastion which will suddenly fall. The danger lies in the risk of an insidious weakening of the castle foundations over a period of time". 178 That risk, in the recusal context, is a reoccurrence of a publicly condemnable conflict of interest between a judge and a party. Furthermore, the foregoing principled analysis belies suggestions that the prevailing judicial recusal regime is sufficient and that reform is unnecessary. IX Saxmere Revisited, Following Reform Following the foregoing analysis of judicial recusal law, it is pertinent to contextualise the mismatch between process theory and the realities of recusal procedure by returning to the Saxmere saga. In doing so, the author looks to substantiate his hypothesis that: 174 Register of Pecuniary Interests Bill 2010 (240–1). See generally: Law Commission Towards a New Courts Act – A register of judges' pecuniary interests?, above n 139; Law Commission Review of The Judicature Act 1908: Towards a New Courts Act, above n 139; Justice and Electoral Committee, above n 139. 176 See generally: New Zealand Bar Association "Submission to the Justice and Electoral Committee on the Register of Pecuniary for Judges Bill 2010"; New Zealand Law Society "Submission to the Justice and Electoral Committee on the Register of Pecuniary for Judges Bill 2010"; Sian Elias CJ (on behalf of the judiciary) "Submission to the Justice and Electoral Committee on the Register of Pecuniary for Judges Bill 2010". 177 Justice and Electoral Committee, above n 139, at 3. 178 Thomas Eichelbaum, former Chief Justice of New Zealand "Judicial Independence Revisited" (Neil Williamson Memorial Lecture, Christchurch, 13 August 1997). 175 44 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD one, a lack of procedural safeguards may have contributed to the controversy; and two, that had procedural safeguards, namely the procedures proposed in this paper, been in place, the controversy could have been mitigated, if not avoided A Disclosure Disclosure, or lack thereof, played a major role in the Saxmere saga. While Wilson J's attempted disclosure, being at the beginning of the proceeding, was timely, it was in private and the details revealed were imprecise. If Wilson J had followed court guidelines, the disclosure should have been made through the court's Registrar. The author posits that the soft nature of the existing disclosure obligations, with its negatively framed duty and heavy emphasis on introspection, may have been a material cause of the minimal disclosure. Beginning with a strong assumption of impartiality from which to introspect whether the Rich Hill business association should be disclosed, Wilson J unilaterally undertook an internal assessment of the interest's relevance and materiality to the proceeding. Notwithstanding that he clearly saw sufficient relevance to attempt disclosure, the method perpetuates the cognitive difficulties he may have faced, like everyone would have, in assessing his own interests – defined earlier as a "systematic inaccuracy caused by unconscious leanings not removable by introspection".179 By starting from a softer assumption of impartiality and shifting the emphasis from introspection to a more positively framed and open disclosure duty, the author opines that the proposed disclosure regime may have led to a more forthright revelation by Wilson J of the interest at first instance. The onus to consider whether a reasonable apprehension of bias arises shifts to the parties, namely Saxmere, rather than remaining a part of Wilson J's introspection. Disclosing in open court would have minimised the uncertainty and negative connotations associated with a private phone call. Further, had the particulars of the business relationship been disclosed, namely that Rich Hill was an active company to which Wilson J and Galbraith had contributed disproportionate levels of capital, the implied waiver of failing to file a recusal motion in a timely manner would have precluded Saxmere's ability to appeal to the Supreme Court on that basis. To emphasise that the Saxmere saga was not a one-off failure by Wilson J to disclose his business relationship with Galbraith, it is helpful to analyse subsequent cases in which the two appeared on the bench and bar, respectively. Of the four cases where this scenario transpired before the Saxmere No 2 appeal was made, only once did Wilson 179 Leubsdorf, above n 140, at 262. 45 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD J disclose to all counsel his business relationship with Galbraith. 180 His rationale for nondisclosure was that he "he did not see it as [a] conflicting [interest]"181 and in any event he was confident that all counsel in the proceedings already knew his business relationship with Galbraith. Without undertaking to validate Wilson J's latter assumption of counsels' knowledge, the author views the former rationale as a personification of the current disclosure duty's frailties. While it is uncertain whether Wilson J would have disclosed more adequately under the proposed disclosure regime, the author hypothesises that at the very least, disclosure would have been fuller than it was under the existing regime. B Filing a Recusal Motion In light of Wilson J's scant disclosure regarding his business relationship with Galbraith, Saxmere may have wanted to inquire further about the relationship to feel more comfortable that it did not raise an apprehension of bias. However, Saxmere may have been hesitant to undertake any additional inquiries or file a recusal motion for fear of annoying Wilson J – which in hindsight may have been likely because Wilson J was clearly of the view that the business relationship did not raise a reasonable apprehension of bias. The existing informal and ad hoc process for raising questions of partiality reasonably invites some trepidation from litigants. Under the proposed regime, making inquiries to better understand a disclosed interest and to guide the decision whether to file a recusal motion will be framed as another common part of civil procedure, similar to interlocutory appeals. Institutionalising this practice should lessen litigants' fears of reprisal. Thus under the proposed regime, Saxmere may have been more inclined to question Wilson J further about the business relationship and subsequently file a recusal motion. This may have minimised, if not eliminated, the subsequent appeals. C An Independent Judge and its Corollary Benefits With more disclosure, Saxmere presumably would have filed a recusal motion considering that its grounds of appeal in Saxmere No 2 were based on imperfect knowledge of the potentially conflicting business relationship. Existing recusal procedure would have left Wilson J to adjudicate the recusal motion. Although he may have discussed the recusal motion with the other two judges on the panel, William Young 180 181 Judicial Conduct Commissioner, above n 11, at 30–31, appendix 1. Judicial Conduct Commissioner, above n 11, at 30–31, appendix 1. 46 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD P and Glazebrook J, the decision would have been his to make. As discussed, judges face cognitive difficulties when self-adjudicating and the prevailing practice is unlikely to foster the public's confidence in an impartial decision being made. Under proposed reform, the recusal motion would have been decided by William Young P and Glazebrook J. The transfer would have facilitated an adversarial process whereby Wilson J could have responded to the motion with his views as to why the business interest does not warrant recusal. Even if William Young P and Glazebrook J arrived at a similar conclusion to Wilson J and dismissed the recusal motion, the public is likely to be more accepting of a decision made by independent judges rather than one made by a challenged judge. Even if the recusal motion was not transferred and was left to be adjudicated by Wilson J, if a comprehensive body of precedent had been available, he would have had more guidance from which to make a decision. Presumably, the precedent would have indicated that the unequal nature of the business relationship and Wilson J's indebtedness to Galbraith warranted a finding similar to that the Supreme Court in Saxmere No 3 came to: that a reasonable apprehension of bias arose and Wilson J should not have sat on the case. Requiring a reasoned decision not only reduces the chance of a judge making a hasty and unconsidered decision to sit or recuse themselves, it builds a body of precedent to better guide judges and litigants alike. Precedent would have been particularly helpful for Wilson J, who, as a judge, was very inexperienced at the time of the case. 182 Evidently, judicial conduct guidelines did not provide the necessary guidance to Wilson J as he had, but did not take, the opportunity to discuss the extent of the potentially conflicting interest with William Young P and Glazebrook J.183 Commentators may postulate that Wilson J's inexperience on the bench played a determinative factor in his conduct. However, his continued failure to disclose the interest in subsequent cases involving Galbraith weakens this theory. Significantly, Wilson J sat on the panel that adjudicated the Muir case which suggests that he would have had intimate knowledge of the law and protocol relating to recusal and yet he failed to adequately apply this knowledge in practice.184 This highlights that despite being well 182 Judicial Conduct Commissioner, above n 11, at [34]. The 2013 Judicial Conduct Guidelines referred to in this paper are a revised version of a set of guidelines that were initially approved by New Zealand judges in 2003. Thus, at the time of Saxmere No 1, Wilson J would have had a set of judicial conduct guidelines at his disposal, as well as the Judges' Benchbook, which is not public available. 184 Muir v Commissioner of Inland Revenue, above n 2. As mentioned, the Supreme Court in Saxmere No 2 affirmed the Court of Appeal's approach in Muir, which brought New Zealand in line with Australia and the United Kingdom's judicial recusal laws. 183 47 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD acquainted with judicial recusal law, its soft and imprecise nature can lead to inconsistent application. The foregoing application of proposed reform to the Saxmere saga illustrates the real remedial potential of proper process in the area of recusal. The parties would have saved a substantial amount of costs that had to be incurred to relitigate the case, not to mention the time delay. Most importantly, the transparent, structured and principled nature of the proposed reform may have averted what the Hon David Parker described as: "a very sad day when we have this taint on our judiciary – and it is a taint. We have to acknowledge it is a taint."185 X Conclusion Judicial impartiality is almost assumed and thus rarely features at the forefront of the legal profession's mind. However, the Saxmere saga thrust it into the limelight with a series of events that culminated in the resignation of a Supreme Court justice: Wilson J. Now that the dust has settled on the controversy and the intense public scrutiny has receded, it is pertinent to question the status quo and investigate the potential cause of the events and, more importantly, whether measures need to be taken to prevent another such occurrence. This paper has adopted a principled approach to analysing judicial recusal law in New Zealand, with a particular focus on procedure. In doing so, a mismatch between process theory and the reality of haphazard self-regulation highlighted the procedural shortcomings of the current recusal paradigm. To remedy this, the author applied aspects of process theory to reform judicial recusal procedure and bring it in line with the general civil litigation process in New Zealand. The proposed reform instils some fundamental practices that are presently absent in recusal procedure. To contextualise the paper's findings, the author revisited the Saxmere saga to: first, posit that a lack of procedural safeguards may have contributed to the saga; and two, to suggest that had the procedural safeguards proposed by this paper been in place, the controversy could have been mitigated, if not avoided. Looking back at Sian Elias CJ's statement that "today respect for courts … has to be earned and re-earned",186 this paper's analysis has questioned judicial recusal law's ability to do just that. To apply the Caesar's wife's principle, "it is not enough that they [judges] give fair, impartial and reasoned judgments. They must bend over backwards to 185 186 (9 November 2010) 668 NZPD 15085. Elias, above n 10. 48 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD be seen to be doing so".187 While the New Zealand judiciary does enjoy the public's confidence, it should not rest on its laurels and view the Saxmere saga as an exception to an otherwise satisfactory judicial recusal regime. It should serve as a wakeup call that significant deficiencies in recusal law exist. In the author's opinion, procedural reform holds considerable promise in providing a robust and principled basis from which judicial impartiality can be – and most importantly, can be seen to be – maintained. Andrew Geddis “No more buddy buddy, no more messing around” (2010) Pundit <http://pundit.co.nz>. The Caesar’s wife principle states that one must be completely above suspicion. 187 49 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD XI Bibliography A Primary Material 1 Cases (a) New Zealand Muir v Commissioner of Inland Revenue [2007] NZCA 334, [2007] 3 NZLR 495. Muir v Judicial Conduct Commissioner [2013] NZHC 3507. Saxmere Co v Wool Board Disestablishment Co [2009] NZSC 72, [2010] 1 NZLR 35 [Saxmere No 2]. Saxmere Co v Wool Board Disestablishment Co [2009] NZSC 122, [2010] 1 NZLR 76 [Saxmere No 3]. Wilson v Attorney-General [2011] 1 NZLR 399. Wool Board Disestablishment Co v Saxmere Co [2007] NZCA 349, [2007] BCL 885 [Saxmere No 1]. (b) Australia Aussie Airlines Pty Ltd v Australian Airlines Pty Ltd [1996] 65 FCR 215, (2006) 135 ALR 753. Ebner v Official Trustee in Bankruptcy (2000) 205 CLR 337. Porter v Magill [2002] 2 AC 357. Re JRL; ex parte CJL (1986) 161 CLR 342. Webb v R (1994) 181 CLR 41. (c) United Kingdom Helow v Secretary of State for the Home Department [2008] 1 WLR 2416. Locabail (UK) Ltd v Bayfield Properties Ltd [2000] QB 451. Metropolitan Properties Co (FGC) v Lannon [1969] 1 QB 577 (CA). 50 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD R v Bow Street Metropolitan Stipendiary Magistrate, ex parte Pinochet Ugarte (No 2) [2000] AC 119. R v Gough [1993] AC 646. R v Sussex Justices, ex parte McCarthy [1924] 1 KB 256. 2 New Zealand Legislation Bill of Rights Act 1990. Constitution Act 1986. Judicature Act 1908. Judicial Conduct Commissioner and Judicial Conduct Panel Act 2004. Oaths and Declarations Act 1957. Sentencing and Parole Reform Act 2010. Supreme Court Act 2003. 3 Bills Register of Pecuniary Interests Bill 2010 (240–1). 4 Parliamentary debates (Hansard) (9 November 2010) 668 NZPD 15085. 5 International material The Universal Declaration of Human Rights, GA Res 217A, III UN GAOR A/810 (1948). B Secondary Material 1 Texts Alexander M Bickel The Lease Dangerous Branch: The Supreme Court at the Bar of Politics (2nd ed, Yale University Press, New Haven, 1986). Grant Hammond Judicial Recusal: Principles, Process and Problems (Hart Publishing, Portland, 2009). Harry Woolf, Jeffrey Jowell and Andrew Le Sueur De Smith's Judicial Review (6th ed, Sweet & Maxwell, London, 2007). 51 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD HP Lee (ed) Judiciaries in Comparative Perspective (Cambridge University Press, Cambridge, 2011). James Boyd White Heracles' Bow: Essays on the Rhetoric and Poetics of the Law (University of Wisconsin Press, Wisconsin, 1985). Jeffrey M Shaman and Jona Goldschmidt Judicial Disqualification: An Empirical Study of Judicial Practices and Attitudes (American Judicature Society, Nashville, 1995). John Locke Second Treatise of Government (Awnsham Churchill, 1690). J Thibaut and L Walker Procedural Justice: A Psychological Analysis (Lawrence Erlbaum Associates, 1975). Kevin Burke and Steve Leben Procedural Fairness: A Key Ingredient in Public Satisfaction (A White Paper of the American Judges Association) (American Judges Association, Williamsburg, 2007). Philip A Joseph Constitutional and Administrative Law in New Zealand (4th ed, Brookers, Wellington, 2014). Richard E Flamm Judicial Disqualification: Recusal and disqualification of Judges (2nd ed, Banks & Jordan Law Publishing Co, Berkley, 2007). Robert H Bork The Tempting of America (Free Press, New York, 1990). Simon Shetreet The Role of Courts in Society (Martinus Nijhoff Publishers, Dordrecht, 1989). 2 Essays in books Gerard McCoy "Judicial Recusal in New Zealand" in HP Lee Judiciaries in Comparative Perspective (Cambridge University Press, 2011) 322. Philip A Joseph "Appointment, discipline and removal of judges in New Zealand" in HP Lee (ed) Judiciaries in Comparative Perspective (Cambridge University Press, Cambridge, 2011) 66. Simon Shetreet "Judging in Society: The Changing Role of Courts" in Simon Shetreet The Role of Courts in Society (Martinus Nijhoff Publishers, Dordrecht, 1989) 467. 52 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD Tom R Tyler "Procedural Justice" in Sarat Austin The Blackwell Companion to Law and Society (Wiley-Blackwell, 2004) 435. 3 Journal articles Amanda Frost "Keeping Up Appearances: A Process-Oriented Approach to Judicial Recusal" (2005) 53(3) Kansas Law Review 531. B Robertson "Judicial Ethics" [2010] 121(4) NZLJ 121. Charles G Geyh "Why Judicial Disqualification Matters. Again" (2011) 30 The Review of Litigation 671. Christopher J Peters "Adjudication as Representation" (1997) 97(2) Columbia Law Review 312. Christopher J Peters "Persuasion: A Model of Majoritarianism as Adjudication" (2001) 96(1) Northwestern Law Review 1. Diane Sivasubramaniam and Larry Heuer "Decision Makers and Decision Recipients: Understanding Disparaties in the Meaning of Fairness" (2007) 44(1) Court Review 62. Emily Pronin, Daniel Lin and Lee Ross "The Bias Blind Spot: Perceptions of Bias in Self Versus Others" (2002) 28 Personality and Social Psychology Bulletin 369. Frank B Cross "Decisionmaking in the US Circuit Court of Appeals" (2003) 91(6) California Law Review 1457. Frederick Schauer "Giving Reasons" (1995) 47 Stanford Law Review 633. Geoffrey C Shaw "HLA Hart's Lost Essay: Discretion and the Legal Process School" (2013) 127(2) Harvard Law Review 666. Jeffrey W Stempel "In Praise of Procedurally Centered Judicial Disqualification – and a Stronger Conception of the Appearance Standard: Better Acknowledging and Adjusting to Cognitive Bias, Spoliation, and Perceptual Realities" (2011) 30 The Review of Litigation 733. John Leubsdorf "Theories of Judging and Judge Disqualification" 62(2) New York University Law Review 237. John M Greacen "Social Science Research on 'Procedural Justice': What are the Implications for Judges and Courts" (2008) 47 Judges' Journal 41. 53 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD John P Frank "Disqualification of Judges" (1947) 56 YLJ 605. John R Allison "Ideology, Prejudgment, and Process Values" (1994) 28 New England Law Review 657. Larry Heuer "What's Just About the Criminal Justice System? Perspective" (2005) 13 Journal of Law & Policy 209. A Psychological Note "Disqualification of a Judge on the Ground of Bias" (1927) 41(1) Harvard Law Review 78. Philip Bryden and Jula Hughes "The Tip of the Iceberg: A Survey of the Philosophy and Practice of Canadian Provincial and Territorial Judges Concerning Judicial Disqualification" 48 Alberta Law Review 569. Robin Cooke "Fundamentals" (1988) NZLJ 158. Saskia Righarts and Mark Henaghan "Public Perceptions of the New Zealand Court System: An Empirical Approach to Law Reform" (2010) 12(2) Otago Law Review 329. Tom R Tyler "Social Justice: Outcome and Procedure" (2000) 35(2) International Journal of Psychology 117. Tom R Tyler "The Role of Perceived Injustice in Defendants' Evaluations of Their Courtroom Experience" (1984) 18(1) Law & Society Review 51. Tom R Tyler "What is Procedural Justice? Criteria Used by Citizens to Assess the Fairness of Legal Procedures" (1988) 22(1) Law & Society Review 103. 4 Internet materials Andrew Geddis “No more buddy buddy, no more messing around” (2010) Pundit <http://pundit.co.nz>. Sensible Sentencing Trust < http://www.sst.org.nz>. 5 "About Us" Sensible Sentencing Trust New Zealand government material "Court of Appeal Recusal Guidelines" (June 2013) Courts of New Zealand <www.courtsofnz.govt.nz>. 54 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD "Guidelines for Judicial Conduct" (March 2013) Courts of New Zealand <www.courtsofnz.govt.nz>. 6 Reports Judicial Conduct Commissioner Decision of the Judicial Conduct Commissioner as to Three Complaints Concerning Justice Wilson (7 May 2010). Justice and Electoral Committee Register of Pecuniary Interests of Judges Bill (21 February 2014). Law Commission Review of The Judicature Act 1908: Towards a New Courts Act (NZLC R126, 2012). Law Commission Towards a New Courts Act – A register of judges' pecuniary interests? (NZLC IP21, 2011). 7 Submissions to the Law Commission and Select Committees Judicial Conduct Commissioner "Submission to the Justice and Electoral Committee on the Register of Pecuniary for Judges Bill 2010". New Zealand Bar Association "Submission to the Justice and Electoral Committee on the Register of Pecuniary for Judges Bill 2010". New Zealand Law Society "Submission to the Justice and Electoral Committee on the Register of Pecuniary for Judges Bill 2010". Sian Elias CJ (on behalf of the judiciary) "Submission to the Justice and Electoral Committee on the Register of Pecuniary for Judges Bill 2010". The Council of Chief Justices of Australia "Guide to Judicial Conduct" (March 2007) Supreme Court of Western Australia <www.supremecourt.wa.gov.au>. WM Wilson QC "Submission to the Law Commission Issues Paper: Towards a new Court Act – A register of judges' pecuniary interests. 8 Media articles Nicky Hager "Appeal Court Judge Faces a Question of Judgement" The Sunday Star Times (New Zealand, 24 August 2008). 55 JUDICIAL RECUSAL IN NEW ZEALAND: LOOKING TO PROCEDURE AS THE PRINCIPLED WAY FORWARD 9 Speeches Philip Havers and Alasdair Henderson "Recent Developments (And Problems) In the Law On Bias" (Hart Judicial Review Conference, The Cavendish Conference Centre, London, 10 December 2010). Sian Elias, Chief Justice of New Zealand "Contemporary Issues for Courts – Demystifying the Judicial Process" (Conference of Chief Justices of Asia and Pacific 2013, Supreme Court, Singapore, 29 October, 2013). Thomas Eichelbaum, former Chief Justice of New Zealand "Judicial Independence Revisited" (Neil Williamson Memorial Lecture, Christchurch, 13 August 1997). 

W4282937673.txt

Chemical Science View Article Online Open Access Article. Published on 05 May 2022. Downloaded on 5/21/2024 1:41:47 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. EDGE ARTICLE Cite this: Chem. Sci., 2022, 13, 6089 All publication charges for this article have been paid for by the Royal Society of Chemistry View Journal | View Issue Direct in situ spectroscopic evidence of the crucial role played by surface oxygen vacancies in the O2sensing mechanism of SnO2† Stefan Kucharski,*ab Pilar Ferrer, c Federica Venturini,c Georg Held, c Alex S. Walton, de Conor Byrne,de James A. Covington,f Sai Kiran Ayyala,f Andrew M. Beale ab and Chris Blackman *a Conductometric gas sensors (CGS) provide a reproducible gas response at a low cost but their operation mechanisms are still not fully understood. In this paper, we elucidate the nature of interactions between SnO2, a common gas-sensitive material, and O2, a ubiquitous gas central to the detection mechanisms of CGS. Using synchrotron radiation, we investigated a working SnO2 sensor under operando conditions via near-ambient pressure (NAP) XPS with simultaneous resistance measurements, and created a depth profile of the variable near-surface stoichiometry of SnO2 x as a function of O2 pressure. Our results reveal a correlation between the dynamically changing surface oxygen vacancies and the resistance Received 25th March 2022 Accepted 4th May 2022 response in SnO2-based CGS. While oxygen adsorbates were observed in this study we conclude that these are an intermediary in oxygen transport between the gas phase and the lattice, and that surface DOI: 10.1039/d2sc01738e rsc.li/chemical-science oxygen vacancies, not the observed oxygen adsorbates, are central to response generation in SnO2based gas sensors. Introduction Conductometric gas sensors (CGS) typically provide a reproducible gas response, have low production and operation costs, and are sold in large quantities across the globe.1 However, CGS typically suffer from limited selectivity and long recovery times.2 The lack of signicant progress in addressing these drawbacks can in part be ascribed to an incomplete picture of atomic interactions during sensing (‘receptor’ function) and their link with the measured macroscopic changes in materials properties (‘transducer’ function).1 Current models of gas sensitivity are based on macroscopic and ex situ spectroscopic measurements.3 The most commonly described is the oxygen ionosorption model, in which monoatomic O adsorbates are produced by dissociative adsorption of gaseous O2, trapping electrons from the conduction band (for an n-type semiconductor) at the material's surface.4–8 The a Department of Chemistry, University College London, 20 Gower St, WC1H 0AJ, London, UK. E-mail: c.blackman@ucl.ac.uk b Research Complex at Harwell, Rutherford Appleton Laboratory, OX11 0FA, Harwell, Didcot, UK c Diamond Light Source, Rutherford Appleton Laboratory, OX11 0FA, Harwell, Didcot, UK d Department of Chemistry, University of Manchester, M13 9PL, Manchester, UK e Photon Science Institute, University of Manchester, M13 9PL, Manchester, UK f School of Engineering, University of Warwick, CV4 7AL, Coventry, UK † Electronic supplementary information https://doi.org/10.1039/d2sc01738e (ESI) available. © 2022 The Author(s). Published by the Royal Society of Chemistry See resulting accumulation of negative surface charge induces an ‘electron depletion layer’ in the material, a volume of decreased Fermi energy (relative to the bulk value in the absence of surface charge). This causes upward band bending (relative to the surface in the absence of adsorbates – the ‘at band’ condition), which is witnessed as increased sensor resistance.9 For the prototype CGS material, SnO2, there is no direct evidence for O species,10 and whilst other adsorbates have been shown to exist on SnO2, for example, O2 , their effect on resistance is considered small.11 Computational studies have been used to provide new insights on the mechanism of interaction between gaseous species and the surface of sensing materials; in general such studies predict that O2 does not interact with stoichiometric surfaces of SnO2, highlighting oxygen vacancies (VO) as essential in enabling adsorption.10,12–16 These studies indicate that gaseous O2 adsorbed on a metal oxide dissociates in a thermally activated process (Fig. 1). However, this does not necessarily lead to the formation of ionosorbed O ; dissociation of O2 across a pair of surface VO will lead to healing of both defects (removing adsorption sites in the process), with no ‘adsorbates’ remaining on the surface (i.e. reversible binding of oxygen at a lattice site).12–14 The consideration of these VO ‘active sites’ signicantly affects the description of band bending at the surface. VO in their neutral state contain two electrons; their formation in the vicinity of the surface introduces donor states below the conduction band minimum in the near-surface region. The Chem. Sci., 2022, 13, 6089–6097 | 6089 View Article Online Open Access Article. Published on 05 May 2022. Downloaded on 5/21/2024 1:41:47 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Chemical Science Edge Article Fig. 1 Visualisation of possible oxygen species adsorbed onto a surface VO on the (110) surface of SnO2. The VO (the locations of absent O atoms) are marked in blue. Reproduced with permission from ref. 12. ionisation of surface VO (e.g. at sufficiently high temperature) must cause accumulation of positive surface charge, inducing a surface ‘electron accumulation layer’, a volume of increased Fermi energy (relative to the bulk value in the absence of surface oxygen vacancies). This causes downward band bending at the surface (relative to the surface in the absence of surface oxygen vacancies – the ‘at band’ condition). Therefore, the healing of VO with increasing oxygen partial pressure is expected to lead to (relatively) upward band bending as a function of decreasing oxygen vacancy concentration. It is worth noting that upward band bending and increased resistance is expected in both ionosorption (increased surface adsorbates) and vacancy modulation (decreased oxygen vacancy concentration) descriptions with increasing oxygen partial pressure, i.e. they are synonymous at the macroscopic scale. Using ex situ X-ray photoelectron spectroscopy measurements (XPS), Semancik et al. have previously inferred a link between decreasing surface VO concentration and increased resistance in SnO2,15 and have also shown that the formation of surface VO results in a surface conductivity layer, and additionally an alternative gas sensing mechanism employing a description of ionised surface oxygen vacancies has recently been proposed.16 Further, Elger and Hess recently published a study correlating changes in the ambient gas composition with surface adsorbed species, inferring changes in oxygen vacancy concentration from UV-vis reection spectra.17 However, to directly link variation in vacancy concentration with change in materials resistance as a function of gas ambient requires a joint macroscopic and spectroscopic picture of a sensor working dynamically under typical temperature and pressure operating conditions. Such ‘operando’ investigations have previously been reported for a variety of spectroscopic techniques.18 Near-ambient pressure (NAP) XPS allows investigation of the chemical state and relative abundance of a material's near-surface atoms through representative spectral peaks in pressures up to several millibars,19 and also provides information on band bending through shis in the binding energy (BE) scale, and consequently it has started to nd application for studying sensors under conditions close to their normal operating environment.20–22 However, to date, there has been no extensive investigation of the most fundamental gas/surface interaction in SnO2 gas sensors, that with O2, and in particular, no in-depth study of oxygen vacancies under dynamic 6090 | Chem. Sci., 2022, 13, 6089–6097 conditions has been presented. In this paper, we use NAP XPS to elucidate the dynamic effect of O2 adsorption on the electronic properties of SnO2 highlighting the role of oxygen vacancies and their interactions with O2 adsorbates in the gas sensing mechanism of SnO2. Results and discussion This investigation aimed to examine oxygen adsorption on polycrystalline SnO2 (as used in the majority of CGS) without extrinsic dopants but with an appreciable density of surface vacancies. Such a surface was achieved through in situ H2 reduction and dehydroxylation immediately prior to the experiments at the ‘VerSoX’ beamline B07 of Diamond Light Source (for details, see ref. 23 and ESI†). This paper presents two experiments: LOW_T, performed at 50  C, and HIGH_T, performed at 350  C. The two temperatures were chosen to represent conditions below and above the 150  C threshold at which, according to ‘ionosorption’ models, diatomic ionosorbed oxygen species (e.g. O2 ) give way to monoatomic ones (e.g. O ).6 Aer establishing a constant temperature, the pressure in the analysis chamber was altered according to the planned experimental steps, denoted as D# (dosing of 1 or 2 mbar O2) and E# (evacuation to UHV). The sensor's resistance was monitored continuously throughout each experiment, including during XPS acquisition, and the XPS measurements were collected aer every pressure change (allowing approximately 1 hour for sample stabilisation); no measurable changes in the resistance were observed as a result of X-ray irradiation of the sensor. The tuneable excitation energy of synchrotron-based XPS was used to examine the spatial distribution of the observed species. The two photon energies used (895 eV and 2000 eV) resulted in sampling depths of approximately 2.5 nm and 5 nm, referred to as ‘shallow’ and ‘deep’, respectively, in this text (see ESI† for depth estimation details). Given the experimental uncertainties, the values of parameters (band bending, O/Sn and ‘O third/Sn’) derived from the XPS spectra reported below have been rounded to the nearest 0.05. Low-temperature experiment (LOW_T) The results of the analysis during LOW_T are presented in Fig. 2. Starting with macroscopic data (bottom panel of Fig. 2), © 2022 The Author(s). Published by the Royal Society of Chemistry View Article Online Open Access Article. Published on 05 May 2022. Downloaded on 5/21/2024 1:41:47 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Edge Article Fig. 2 Results of phenomenological (bottom) and XPS (middle and top) investigation during experiment LOW_T. The ‘shallow’ and ‘deep’ plots present quantification of XPS spectra with 2.5 and 5 nm sampling depth, respectively. Sn p.pos denotes the binding energy position of the Sn 3d5/2 peak. the resistance of the sensor aer reduction (indicated on the gure as ‘UHV before’) stabilised around 300 U (for details on the reduction procedure, see ESI†). As soon as O2 was introduced (D1), the resistance increased rapidly up to 4.2 kU before immediately starting to decrease to 640 U. This phenomenon happened before the O2 pressure reached the maximum value (see Fig. S8 in ESI†) and therefore is not linked to the observed overshoot in O2 pressure and subsequent stabilisation at 1 mbar. This initial ‘peak-type’ response was followed by a gradual, small increase and stabilisation of the resistance at a value slightly larger than the initial one, about 750 U (response 2.5, Rpresent/Rprevious). Notably, the sensor's resistance was insensitive to the subsequent O2 evacuation (E1), and there was no decrease in resistance upon evacuation during any of the E# steps. Each following oxygen exposure aer D1 had a smaller effect on both the ‘peak’ and ‘gradual’ response; aer stabilisation in D2, the resistance was around 850 U (an increase of 100 U compared to D1, i.e. a response of 1.1) and aer stabilisation in D3, the resistance was at the same value as in D2 (850 U, i.e. a response of 1.0). Together, these results indicate that, from the macroscopic point of view, the changes occurring at the surface of SnO2 upon exposure to O2 at low temperature (50  C) are largely irreversible. The limited change in resistance observed from D1 onwards means that the microscopic processes occurring here cannot be the ones responsible for the high sensitivity of SnO2-based sensors (the invariance of resistance measurements over an extended time period for each dose suggests the limited change in resistance is not simply © 2022 The Author(s). Published by the Royal Society of Chemistry Chemical Science a function of slow kinetics but is indicative of a ‘saturated’ response). Changes in the corresponding XPS spectra were used to understand the microscopic processes occurring at the sensor's surface during each dose and evacuation step. The Sn 3d spectra collected during LOW_T (Fig. 3) showed no changes in the peak shape resulting from O2 exposure. The well-dened shape of the Sn peaks allows reliable estimation of their position and, in turn, band bending analysis, which is presented in Fig. 2 (middle panel, Sn p.pos). Aer reaching the experimental temperature (‘UHV before’), the ‘shallow’ Sn 3d peak appeared at 487.00 eV, and no O2 pressure-dependent changes in its position were observed irrespective of the dose or evacuation state. Likewise, in the ‘deep’ spectra, no peak shis were discernible beyond experimental uncertainty. Therefore we conclude that on exposure of the sensor to O2 at 50  C there is no observable band bending. Despite the fact that no band bending was observed, the dosing of O2 signicantly changed the peak shape of the O 1s photoemission. In order to assess these changes quantitatively, the peak was tted with two components (for details, see ESI†). The rst component (denoted ‘O lattice’ in Fig. 3) corresponds to the O atoms constituting SnO2 and was tted without any constraints in its position, FWHM or area. Aer introducing O2 (D1), another peak was required to obtain a good t, corresponding to newly-formed oxygen species, arbitrarily denoted Fig. 3 Comparison of Sn 3d and O 1s high-resolution spectra collected during steps ‘UHV before’, D1 and E1 of experiment LOW_T. Peaks were offset along the Y-axis. The lower signal intensity of Sn during D1 compared to the UHV steps is attributable to the attenuation of photoelectrons by the O2 gas present during that step. The O 1s peaks were normalised with respect to their corresponding Sn 3d peak areas. Chem. Sci., 2022, 13, 6089–6097 | 6091 View Article Online Open Access Article. Published on 05 May 2022. Downloaded on 5/21/2024 1:41:47 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Chemical Science as ‘O third’ to indicate that they are not related to the SnO2 lattice, i.e. ‘third-party species’. The ‘O third’ component parameters (position, FWHM and area) were also le unconstrained, and their magnitude is a measure of the difference between the shapes of the ‘O lattice’ component and the observed photoemission. Similar peak tting was performed at every step of the investigation for the ‘shallow’ and ‘deep’ spectra, allowing estimation of the relative abundance and distribution of ‘O lattice’ and ‘O third’, reported as O/Sn and ‘O third’/Sn ratios. The quantication described above is presented in Fig. 2 over multiple dose and evacuation steps and shows that the ‘O third’/Sn ratio increased during D1 (1 mbar O2) from approximately 0 during ‘UHV before’ for both ‘shallow’ and ‘deep’ spectra to 0.50 for shallow spectra (middle panel) and 0.15 for deep spectra (top panel). Subsequently, neither the removal (E#) nor reintroduction (D#) of O2 inuenced the ‘O third’ peak intensity, which remained constant within experimental precision. The larger increase in the ‘shallow’ (0.50) compared to ‘deep’ (0.15) spectra indicates that the new oxygen species are localised close to the surface. However, relatively little information about the surface species’ chemical identity can be extracted from these spectra (Fig. 3). The position of ‘O third’, ca. 532.10 eV, falls within a binding energy region characteristic for many species, including adventitious organic contamination, surface hydroxyls and adsorbed water, as well as for oxygen adsorbates.20,24 Therefore, the identity of the species present cannot be determined from the binding energy of the peak alone but has to be deduced via exclusion analysis. Considering the high purity O2 used in this experiment (N6.0, H2 # 0.05 ppm, H2O # 0.5 ppm) and the mismatch between trends observed in ‘O third’ and C 1s spectra (which, if correlated, would point to the ‘O third’ emission originating from organic contamination), it is concluded that the species appearing at the surface were not related to surface hydroxylation or to adventitious organic contamination. For detailed considerations, see ESI.† Since other possibilities were excluded, the ‘O third’ peak must be ascribed to some form of adsorbed oxygen species. However, these are unlikely to be the O species invoked in the ionosorption model of gas sensing, as the surface density of such species is limited to 10 3–10 5 of a monolayer.11 Assuming that only the outermost surface atoms contribute to the signal (3 O and 2 Sn atoms per (110) surface unit cell) and the maximum adsorbate density (10 3 ML; 2 O per unit cell is 1 ML), the O adsorbate peak should be no larger than 0.13% of the lattice oxygen peak area, and even smaller when all lattice atoms in the sampling depth are considered. This is much less than the area of ‘O third’ relative to ‘O lattice’ (35%) and below the detection limit in XPS.25 Therefore, given their large relative abundance the species must be uncharged with respect to the lattice and therefore likely to be diatomic and covalently bound onto surface VO (i.e. species identied as O2-stand and O2-lie in Fig. 1). Aer the initial increase between ‘UHV before’ and D1, indicating the appearance of adsorbates, the ‘O third’/Sn ratio (Fig. 2) did not decrease signicantly upon evacuation in any of the following E# steps. This means that at 50  C adsorption is 6092 | Chem. Sci., 2022, 13, 6089–6097 Edge Article irreversible, and the adsorption sites are not recovered in UHV. Since the ratio reaches its plateau value during D1, it can be assumed that nearly all active adsorption sites are occupied at this point; consequently, the lack of additional available sites prevents the adsorption of new dioxygen molecules during D2 and D3. Finally, the formation of these oxygen adsorbates does not inuence band bending, as the Sn p.pos does not change when ‘O third’ emerges (Fig. 2, middle panel). Therefore, these adsorbates are not connected to the sensor's response as normally described under the framework of ionosorption theory, and other mechanisms for resistance change must be at play. The area of the ‘O lattice’ component (Fig. 3) divided by the corresponding Sn 3d peaks, denoted as O/Sn ratio, determines the SnO2 x lattice stoichiometry and therefore measures the VO density in the analysed volume. In the ‘shallow’ spectra (Fig. 2, middle panel), the O/Sn ratio was approximately 1.35 aer reduction (‘UHV before’). During steps D1 and E1, the ratio increased to 1.50 to 1.60 and subsequently remained invariant within experimental precision (note these values are calculated using standard relative sensitivity factors and therefore, whilst comparable with one another, do not represent the true Sn:O stoichiometry). At the same time, the O/Sn ratio derived from ‘deep’ spectra (Fig. 2, top panel) showed a steady, gradual increase from 1.50 to 1.60 throughout the experiment and, considering the uncertainties, is barely noticeable. Since this parameter determines the SnO2 stoichiometry in the analysed volume, an increase can be interpreted as a measure of oxygen vacancy healing via an inux of oxygen into the lattice. The larger variation in ‘shallow’ O/Sn suggests that the stoichiometry change is primarily occurring very close to the surface, consistent with dynamic exchange of oxygen atoms with surface vacancies. Given that the changes in the ‘shallow’ volume will also affect the ‘deep’ spectra, and considering the low rate of VO hopping in SnO2 at 50  C (very slow diffusion),26,27 it is likely that the ‘deep’ O/Sn increase is entirely due to the near-surface stoichiometry change observed in ‘shallow’ spectra. These trends in the O/Sn ratio correlate with the small, gradual overall increase in resistance observed during LOW_T. The largest increase in both the O/Sn ratio (1.35 to 1.50, Fig. 2, middle panel) and the resistance (300 U to 750 U, Fig. 2, bottom panel) is observed (for measurements made in UHV) aer the rst O2 exposure (‘UHV before’ vs. E1). Subsequently, both O/Sn and resistance increase less between E1 and E2 and negligibly between E2 and E3. Moreover, the temperature of 50  C is considered too low to enable spontaneous surface reduction; therefore, neither the resistance nor the O/Sn ratio decrease in UHV but progress towards their equilibrium values determined by the oxygen pressure during exposure. To try and describe the features seen in LOW_T, we refer to the model by Yang et al. (Fig. 1). We conjecture that aer H2 reduction and dehydroxylation, the surface is vacancy rich (low O/Sn ratio), and on initial O2 exposure, the O2-stand conguration of adsorbed oxygen is obtained (‘O third’), causing the initial peak change in resistance. Subsequently, the O2-stand is converted to the O2-lie conguration, correlating with the resistance drop immediately aer the peak. This is not entirely consistent with the description of Yang, as the partial charge © 2022 The Author(s). Published by the Royal Society of Chemistry View Article Online Open Access Article. Published on 05 May 2022. Downloaded on 5/21/2024 1:41:47 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Edge Article (charge transferred from SnO2 to O2) of O2-lie is calculated to be greater than for O2-stand, however a change in nature of adsorbed species such as this would account for the resistance prole observed. These surface species are clearly stable at 50  C as exposure to UHV does not cause the species to desorb (cycling between D# and E# does not change the ‘O third’/Sn ratio). We also conjecture that at this temperature (compared to 350  C, see below), there is not enough energy to overcome the barrier to dissociate all the adsorbed oxygen fully (Fig. 1). The dissociation of O2 corresponds to the maximum charge transfer observed by Yang and hence is expected to correspond to a large change in sensor resistance. However, this provides an inconsistent piece of data – the change in ‘lattice oxygen’ (O/Sn) at 50  C is signicant (1.35 to 1.50) but does not correlate with signicant resistance change (compared to 350  C, see Fig. 4 and discussion below). Referring to the recently described surface vacancy description of gas sensor response,16 a potential explanation for this lack of resistance change is that the number of ionised near-surface oxygen vacancies increases with increasing temperature;28 only ionised VO are expected to contribute to resistance change (non-ionised neutral VO already localise electrons at the vacancy site). This would also account for the lack of band bending observed even though the vacancy concentration appears to vary signicantly. In summary of the LOW_T experiment, we observed a substantial amount of oxygen adsorbates which, due to the lack of corresponding band bending, are likely diatomic and not (formally) charged (see Fig. 1), and not directly related to the response generation in SnO2. At this temperature, the O2 adsorption is irreversible since ‘O third’ does not decrease in Chemical Science UHV. Moreover, the lack of bend bending and minimal resistance change with increasing surface stoichiometry, as presented in Fig. 4, suggest that adsorbate dissociation does not localise electrons onto non-ionised vacancies.16 Additionally, given that the low temperature prevents vacancy diffusion from the bulk towards the surface, any vacancies healed here must be limited to the surface-most atomic layer, which is reected in the signicant difference between ‘shallow’ and ‘deep’ O/Sn variance. Finally, the link between the observed decrease in vacancy density and baseline resistance increase through ‘gradual’ response is consistent with other studies, in which changing oxygen vacancy concentration was correlated with sensor dri.26 High-temperature experiment (HIGH_T) The results of the HIGH_T experiment, analogous to LOW_T but performed at 350  C, are presented in Fig. 5. Similar to LOW_T, the lowest resistance (120 U, ‘UHV before’) was observed directly aer reduction (for details, see ESI†). During D1, the resistance increased quickly to 2.5 kU and then gradually to 4.5 kU (response 38, substantially higher than the LOW_T response of 2.5). The resistance measurements during E1 and E2 (UHV) are considered anomalous as resistance increased on evacuation, and therefore they have been greyed out in Fig. 5 (see ESI†). This behaviour was observed during the live experiment, and consequently, the O2 dose pressure was increased to 2 mbar (D3.5, D4.5), at which point the sensor started behaving as expected, i.e. resistance decreased on evacuation to UHV. The pressure increase during D3.5 caused the resistance to rise to Fig. 4 Summary of band bending (Sn 3d p.pos) and resistance change as a function of oxygen vacancy density in the near surface region derived from the LOW_T and HIGH_T experiments. In the LOW_T plot, the red resistance data points correspond to E# steps. In the HIGH_T plot, the ‘anomalous’ resistance data were omitted; for details, see the discussion of the HIGH_T experiment below and ESI†. © 2022 The Author(s). Published by the Royal Society of Chemistry Fig. 5 Results of phenomenological (bottom) and XPS (middle and top) investigation during experiment HIGH_T. The ‘shallow’ and ‘deep’ plots present quantification of XPS spectra with 2.5 and 5 nm sampling depth, respectively. Sn p.pos denotes the binding energy position of the Sn 3d5/2 peak. Chem. Sci., 2022, 13, 6089–6097 | 6093 View Article Online Open Access Article. Published on 05 May 2022. Downloaded on 5/21/2024 1:41:47 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Chemical Science approximately 7 kU; however, unlike in LOW_T, the subsequent removal of O2 (E3) resulted in a pronounced decrease in resistance (initially down to 550 U although later only to 2.3 kU once the resistance value stabilised). Reproducible changes in resistance were observed during subsequent steps, with the resistance increasing to around 6–7 kU during exposure to O2 (D4, D4.5 and D5) and decreasing to 3.5 kU aer stabilisation in E4 (Fig. 5). The ‘shallow’ band bending analysis (Sn p.pos, Fig. 5, middle panel) is derived from the Sn 3d spectra shown in Fig. 6. Signicant oxygen pressure-dependent changes in band bending were observed during HIGH_T in the ‘shallow’ spectra, in contrast to LOW_T (Fig. 2) where negligible band bending was observed. In D1, the Sn peak shis from 487.10 eV to 486.75 eV due to the bands bending upwards by over 0.30 eV. The subsequent O2 removal (E1) caused unbending of the bands; however, the Sn p.pos did not revert to its original value of 487.10 eV, but only to 486.95 eV, a value closer to that before reduction (487.00 eV). This value became the new maximum, to which the peak returned during subsequent E# steps. During the D# steps, the Sn p.pos consistently shied to 486.70 eV ( 0.25 eV), apart from D3.5 and D4.5, during which it was slightly lower at around 486.65 eV ( 0.30 eV), correlating with the higher O2 pressure used during these steps. Similar trends were observed in ‘deep’ spectra (Fig. 5, top panel), although the values were less consistent due to the larger uncertainty associated with higher photon energy used to obtain the data (see band bending discussion in LOW_T). Additionally, the Fig. 6 Comparison of Sn 3d and O 1s high-resolution spectra collected during steps ‘UHV before’, D1 and E1 of experiment HIGH_T. Peaks were offset along the Y-axis. The lower signal intensity of Sn during D1 compared to the UHV steps is attributable to the attenuation of photoelectrons by the O2 gas present during that step. The O 1s peaks were normalised with respect to their corresponding Sn 3d peak areas. 6094 | Chem. Sci., 2022, 13, 6089–6097 Edge Article difference between Sn p.pos during D# and E# was smaller in ‘deep’ spectra, varying by only about 0.20 eV (between 486.80 and 487.00 eV) as opposed to 0.30 eV in ‘shallow’ spectra. This is consistent with the bands bending more strongly close to the surface, with the ‘deep’ Sn 3d peak position inuenced more by the constant bulk Fermi level and the ‘shallow’ peak inuenced more by the surface Fermi energy pinning. The O/Sn ratio also showed remarkable reversibility in the ‘shallow’ spectra (Fig. 5, middle panel), as seen for band bending. Starting at an O/Sn value of 1.35 aer reduction (the same as seen in LOW_T), the ratio increased signicantly in D1 to about 1.70 (1.50 in LOW_T). Unlike LOW_T, O2 evacuation (E1) resulted in a decrease in O/Sn stoichiometry (beyond experimental uncertainty), down to 1.55 (i.e. not as ‘reduced’ as the starting point aer H2 reduction). The O/Sn values of 1.70 for D# and 1.55 for E# were then consistently reproduced in all steps, except D3.5 and D4.5, where the O/Sn ratio was slightly larger (approximately 1.80), consistent with the increased O2 pressure (although it is worth noting that the D3.5 and D4.5 values carry a larger uncertainty in the O/Sn ratio due to the increased O2 pressure during data collection and the increased uncertainty in the required signal attenuation calibration, see ESI†). Although similar trends were observed in the ‘deep’ O/Sn ratio (Fig. 5, top panel), the changes were less pronounced, similar to observations in LOW_T, with the initial O/Sn value of about 1.50 (‘UHV before’) increasing to 1.70 on oxygen dosing (D1). Upon removal of O2, the O/Sn value in the deep spectra decreased by about 0.05 and oscillated between 1.70–1.75 during D# steps and 1.65–1.70 during E# steps. The error bars associated with these values are signicantly overlapping, indicating that the observed changes (apart from the initial increase in D1) are within uncertainty. The difference between the lattice O/Sn ratio in ‘shallow’ and ‘deep’ spectra (middle and top panels respectively, Fig. 5) under UHV conditions suggests that oxygen vacancies are dynamically formed mainly close to the surface. In the ‘shallow’ spectra, the O/Sn ratio increased by 26% (1.70/1.35) upon O2 exposure in step D1. This same change resulted in only a 13% (1.70/1.50) increase in the ‘deep’ O/Sn ratio. Subsequent O2 evacuation in E1 caused a 9% (1.55/1.70) decrease in the ‘shallow’ spectra and only about 3% (1.65/1.70) in the ‘deep’. If the VO were distributed homogenously beyond the thickness of the ‘shallow’ analysis volume, identical changes would be expected in both sets of spectra, hence it can be concluded that the VO (healed or produced, depending on the conditions) constituted a more signicant fraction of the lattice sites at the surface. The variable density of near-surface oxygen vacancies, which reects changes in the O2 pressure, is correlated with the trends in both the band bending (Sn p.pos) and sensor resistance, as shown in Fig. 4 (HIGH_T). As the density of vacancies decreases due to the incorporation of gaseous O2 into the lattice, the bands bend upwards (Sn p.pos decreases) and the resistance of the sensor increases. The changes are reproducible, since the repeated exposure to O2 and evacuation to UHV return the system to a similar VO density, band bending and resistance as during the previous analogous steps, thus allowing distinction © 2022 The Author(s). Published by the Royal Society of Chemistry View Article Online Open Access Article. Published on 05 May 2022. Downloaded on 5/21/2024 1:41:47 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Edge Article of separate regions on the plot corresponding to the E#, D# and D#.5 steps (in addition to the ‘UHV before’ which can only be attained through in situ reduction in H2). The close correlation between these three parameters indicate that the charge carrier concentration, which determines the Fermi level position and hence observable band bending and sensor's conductivity, depends on the density of surface vacancies, i.e. the vacancies act as electron donors, directly determining the sensor's resistance. A conductivity model based on vacancy healing and formation was recently proposed for SnO2-based CGS by Sun and Lu, which allowed them to model the conduction electron concentration and sensor response in both the ‘electron depletion’ and ‘electron accumulation’ regime depending on the density of near-surface vacancies relative to the bulk value.29 Our direct in situ spectroscopic evidence of the correlation between near-surface vacancy density and sensor's resistance and band bending fully supports such a mechanism. Whereas an abundance of adsorbates was observed in the XPS spectra during LOW_T (Fig. 3), the sensor's exposure to O2 in HIGH_T has relatively little effect on the shape of the O 1s photoemission spectra; Fig. 6 shows the changes in ‘shallow’ O 1s peaks between ‘UHV before’, D1 and E1. The binding energy position of the lattice component shis visibly aer O2 exposure (‘UHV before’ vs. D1 and E1), which is also visible in the Sn 3d peaks, and is indicative of band bending. Again, while a single component (‘O lattice’) is sufficient to t the observed photoemission of ‘UHV before’, another component, ‘O third’, is required in the case of D1. However, the magnitude of this component (‘O third’/Sn ratio of 0.10) is only 6% of the lattice peak, which is relatively small compared to the analogous peaks in LOW_T (35%). Given that the peak appears as a result of O2 exposure and at the same binding energy as during the LOW_T experiment, ca. 532.10 eV, again consistent with the ‘O third’ species being oxygen adsorbates,24 it was therefore assigned to diatomic (formally) neutral oxygen adsorbed onto surface oxygen vacancies, such as the O2-stand and O2-lie species shown in Fig. 1. Notably, in contrast to LOW_T, the ‘O third’ emission for HIGH_T disappears aer evacuation (E1 peak in Fig. 5), i.e. fully reversible formation of oxygen adsorbates is observed. As shown in Fig. 5 (‘O third’/Sn), the component reappears in all subsequent D# steps at a similar intensity and is removed during the following E# steps (‘O third’/Sn oscillates between circa 0.10 for D# and 0 for E#). While the change in ‘O third’ between D# and E# steps is evident in the ‘shallow’ spectra, the ‘deep’ spectra show much smaller variation (the error bars of data points overlap signicantly, and there are no clear trends visible). Although the amount of detected oxygen adsorbates (‘O third’) in HIGH_T is substantially smaller than in LOW_T, it is still well above the theoretical maximum for charged adsorbates (Weisz limit, ‘O third’/‘O lattice’ # 0.13%). Therefore, these spectroscopically observed species are unlikely to be O , even though the temperature of this experiment has previously been associated with O2 dissociation into charged monoatomic species.3 The difference in ‘O third’ intensity between the LOW_T and HIGH_T experiments can be rationalised in terms of two factors. Firstly, the elevated temperatures cause increased © 2022 The Author(s). Published by the Royal Society of Chemistry Chemical Science dissociation of the adsorbed dioxygen, healing one or two oxygen vacancies and removing adsorption sites in the process.15 Supporting this notion is the observed increase in the O/Sn ratio during O2 exposures (D# in Fig. 5), more pronounced in the ‘shallow’ spectra, indicating surface vacancy healing and (coincidentally) removing some adsorption sites. Secondly, since the ‘O third’ peak disappears in UHV, it can be concluded that the adsorption of O2 is reversible at this temperature, in agreement with TPD and ESR studies which indicate that diatomic oxygen desorbs from the surface by around 100– 150  C,11 suggesting a more dynamic occupation of available adsorption sites than at low temperature. Although we cannot exclude the presence of low concentrations of charged oxygen adsorbates below the detection limit of our experiment, it is clear that the surface oxygen vacancy concentration changes very signicantly when cycling between UHV and 1 mbar O2 at 350  C (around a 25% change in surface lattice oxygen concentration – O/Sn ratio). These changes in VO concentration (Fig. 7, O/Sn) must be expected to exert a large inuence on the electronic structure (charge distribution) in the near-surface region (assuming surface VO are ionised at the given temperature) and are correlated with both band bending and sensor response (Fig. 7) as a function of O2 pressure.16 Application of ‘Occam's Razor’ therefore leads us to conclude that variable surface vacancy concentration is responsible for the O2 sensing mechanism of SnO2, not extrinsic charged oxygen species. A similar conclusion was reached by Andrae et al. who used synchrotron NAP XPS to examine the interaction of O2 with donor-doped SrTiO3 to interpret (non-simultaneous) resistance measurements.30 There, the actor for band bending (and hence resistance change) was also concluded not to be due to charged oxygen adsorbates, but was due to (increasing) formation of a depletion layer (q.v. decreasing formation of an accumulation layer) with increasing O2 partial pressure, resulting from formation of negatively charged strontium cation vacancies (q.v. positively charged oxygen anion Fig. 7 Correlation between O/Sn ratio of the lattice component (VO concentration), Sn p.pos relative to ‘UHV before’ (band bending) and the sensor response under dynamically changing O2 pressure conditions during final stages of experiment HIGH_T. The initial steps were omitted due to the anomalous resistance measurements recorded during steps E1 and E2. Chem. Sci., 2022, 13, 6089–6097 | 6095 View Article Online Chemical Science vacancies) in the SrTiO3 lattice due to precipitation of phaseseparated SrO at the surface. Our results are also qualitatively consistent with the results of Semancik et al., who previously correlated upward band bending with surface oxidation and vacancy healing.31 Open Access Article. Published on 05 May 2022. Downloaded on 5/21/2024 1:41:47 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Conclusions In this study, joint macro- and spectroscopic analyses were performed using synchrotron NAP XPS with simultaneous resistance measurements on sensors exposed to O2 and UHV at both room and elevated temperatures. The results of this study show that exposure of (in situ reduced) sensors to O2 at elevated temperatures results in resistance and band bending changes, typical of SnO2-based sensors operation. These parameters are correlated with signicant changes in the near-surface oxygen vacancy concentration under dynamic pressure conditions and we conclusively show that the adsorbed oxygen species observed upon O2 dosing are not responsible for the measured resistance change. The decrease in surface oxygen vacancy concentration on O2 dosing must lead to a decrease in positive charge in the near-surface region of SnO2, and hence upward band bending, and therefore we see no need to invoke additional entities (e.g. negatively charged ‘ionosorbed’ oxygen) to describe the same phenomenon. Consequently, we conclude that the resistance change in SnO2 sensors in response to O2 does not result from extrinsic negatively charged surface oxygen species but rather from the dynamic exchange of lattice oxygen with the gas ambient via the formation and healing of surface oxygen vacancies. This challenges the current unproven conceptions in the eld of gas sensing8 but is fully consistent with the recent work in the eld of solid-state physics on vacancy-induced surface conductivity layers in metal oxides.16 Understanding the importance of oxygen vacancies provides rules for targeted design of higher performance (n-type) gas sensing materials via the deliberate manipulation of surface oxygen vacancy concentration, for instance by surface modication with high valent oxyphilic species.32 The method we developed can be readily applied to study a range of sensor materials under exposure to various gases, which we have demonstrated can provide new fundamental understanding of the surface chemistry in CGS. In light of the evidence presented in this paper, we believe such models represent a promising direction towards a complete understanding of CGS operation and hence their performance enhancement. Data availability The datasets supporting this article have been uploaded as part of the ESI.† Author contributions S. K.: conceptualization, data curation, formal analysis, investigation, methodology, visualisation, writing – original dra; C. Blackman: conceptualization, formal analysis, funding acquisition, project administration, supervision, writing – original 6096 | Chem. Sci., 2022, 13, 6089–6097 Edge Article dra; P. F.: conceptualization, investigation; G. H. and A. S. W.: conceptualization, writing – review & editing; C. Byrne.: conceptualization, methodology; F. V.: conceptualization; J. A. C. and S. K. A.: resources; A. M. B.: writing – review & editing. Conflicts of interest There are no conicts to declare. Acknowledgements This work was supported by the Engineering and Physical Sciences Research Council (EP/R512400/1). We acknowledge Diamond Light Source for time on Beamline B07-C ‘VerSoX’ under Proposal SI-25766. Notes and references 1 N. Barsan, D. Koziej and U. 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Chen and J. Hu, J. Phys. Chem. C, 2014, 118, 28548–28561. 15 D. F. Cox, T. B. Fryberger and S. Semancik, Phys. Rev. B: Condens. Matter Mater. Phys., 1988, 38, 2072–2083. 16 C. Blackman, ACS Sens., 2021, 6, 3509–3516. 17 A. Elger and C. Hess, Angew. Chem., Int. Ed., 2019, 58, 15057– 15061. 18 A. Gurlo and R. Riedel, Angew. Chem., Int. Ed., 2007, 46, 3826–3848. 19 D. E. Starr, Z. Liu, M. Hävecker, A. Knop-Gericke and H. Bluhm, Chem. Soc. Rev., 2013, 42, 5833. 20 M. Vorokhta, I. Khalakhan, M. Vondráček, D. Tomeček, M. Vorokhta, E. Marešová, J. Nováková, J. Vlček, P. Fitl, M. Novotný, P. Hozák, J. Lančok, M. Vrňata, I. Matolı́nová and V. Matolı́n, Surf. Sci., 2018, 677, 284–290. 21 P. Hozák, M. Vorokhta, I. Khalakhan, K. Jarkovská, J. Cibulková, P. Fitl, J. Vlček, J. Fara, D. Tomeček, © 2022 The Author(s). Published by the Royal Society of Chemistry View Article Online Edge Article 22 Open Access Article. Published on 05 May 2022. Downloaded on 5/21/2024 1:41:47 PM. 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Journal of Modern Physics, 2020, 11, 920-927 https://www.scirp.org/journal/jmp ISSN Online: 2153-120X ISSN Print: 2153-1196 Quantization of Newton’s Gravity Mario C. Rocca1,2,3*, Angelo Plastino1,3,4 Departamento de Física, Universidad Nacional de La Plata, La Plata, Argentina Departamento de Matemática, Universidad Nacional de La Plata, La Plata, Argentina 3 Consejo Nacional de Investigaciones Científicas y Tecnológicas (IFLP-CCT-CONICET), La Plata, Argentina 4 SThAR-EPFL, Lausanne, Switzerland 1 2 How to cite this paper: Rocca, M.C. and Plastino, A. (2020) Quantization of Newton’s Gravity. Journal of Modern Physics, 11, 920-927. https://doi.org/10.4236/jmp.2020.116056 Received: May 25, 2020 Accepted: June 14, 2020 Published: June 17, 2020 Copyright © 2020 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access Abstract In this work we will use a recently developed non relativistic (NR) quantization methodology that successfully overcomes troubles with infinities that plague non-renormalizable quantum field theories (QFTs). The ensuing methodology is here applied to Newton’s gravitation potential. We employ here the concomitant mathematical apparatus to formulate the NR QFT discussed in the well known classical text-book by Fetter and Walecka. We emphasize the fact that we speak of non relativistic QFT. This is so because we appeal to Newton’s gravitational potential, while in a relativistic QFT one does not employ potentials. Our main protagonist is the notion of propagator. This notion is of the essence in non relativistic quantum field theory (NR-QFT). Indeed, propagators are indispensable tools for both nuclear physics and condensed matter theory, among other disciplines. In the present work we deal with propagators for both fermions and bosons. Keywords Non-Relativistic Quantum Field Theory, Newton’s Gravity, Schwartz’ Distributions 1. Introduction 1.1. Preliminaries In this work we will use a recently developed non relativistic quantization methodology that successfully overcomes all troubles of non-renormalizable QFT [1]. The essential result of such procedures is that we can dispense with renormalization and counter-terms. The reader can consult the recent references [1] [2] [3] [4] [5]. The proofs given there are conclusive. DOI: 10.4236/jmp.2020.116056 Jun. 17, 2020 920 Journal of Modern Physics M. C. Rocca, A. Plastino The above claims are validated because infinities in Feynman diagrams, that arise in the convolution of quantum propagators (QP), disappear if one 1) represents QP by ultra-hyperfinctions (a generalization of Schwartz’ distributions) and follows this technique with an appropriate Laurent expansion. The facts 1) and 2) above are clearly explained, with all kind of details, in [1] [2] [3] [4] [5]. Accordingly, no more mathematical aspects of the procedure need to be given in this paper. The techniques of [1] [2] [3] [4] [5] are here applied to Newton’s gravitation potential. We strongly emphasize the fact that, since we will be inserting a gravitational potential into a Schrödinger Equation (SE), the ensuing discussion is per force non-relativistic, and as such is the character of SE. 1.2. Organizing Our Material In Section 2 we revisit Newton’s gravity. Section 3 is devoted to an explicit display of results belonging to [6], concerning non relativistic quantum field theory (NR-QFT). In Section 4 we apply the results of Sections 2 and 3 so as to obtain the N-QFT of Newton’s gravity. We discuss, as examples, the calculation of the self-energy for fermions and of the dressed propagator for both, bosons and fermions, to first order in perturbation theory. Some conclusions are drawn in Section 5. 2. Newton’s Gravity As stated above, r −1 is viewed here as 1 r −= 1 −1 −1  PV 1 . ( r − i0 ) + ( r + i0 ) =  2 r (2.1) Remember also that δ ( r ) = 0. (2.2) We need now the Fourier transform of r −1 . We have ∫r −1 ik ⋅ x e d3 x  ∞ π2 2 π  ∞ π 2π   i ( k + i ) r cos θ i ( k − i ) r cos θ = lim  ∫ ∫ ∫ e r sin θ drdθ dφ + ∫ ∫ ∫ e r sin θ drdθ dφ  .  →0 0 π 0  0 0 0  2 (2.3) Integrating over φ one finds  ∞ π2  ∞π   i ( k + i ) r cos θ i ( k − i ) r cos θ lim  2π ∫ ∫ e r sin θ drdθ + 2π ∫ ∫ e r sin θ drdθ  .  →0 0 π  0 0  2 (2.4) Evaluating now for θ we reach i k − i r ∞  ei ( k + i ) r e ( )   lim 2π  ∫  −  dr  .  →0  0  i ( k + i ) i ( k − i )   (2.5) Finally, dealing with the variable r we arrive at DOI: 10.4236/jmp.2020.116056 921 Journal of Modern Physics M. C. Rocca, A. Plastino  1  1 + 2π lim  . 2 2  →0  ( k + i ) ( k − i )  (2.6) As an example, consider now the anti transform of 4πk −2 and verify that it is 1 PV . r   1 1 1 2π  +  =4πPV 2 ≡ 4πk −2 . 2 2 k  ( k + i 0 ) ( k − i 0 )  (2.7) One has 4π ( 2π ) 3 ∫k −2 − ik ⋅ x 3   1 = lim  2  → 0 2π  e π ∞2 dk ∫∫e − i ( r − i ) k cos θ 0 0 1 sin θ dkdθ + 2 2π ∞π ∫ ∫π e − i ( r + i ) k cos θ 0 2   sin θ dkdθ  ,  (2.8) so that − i r − i k i r + i k 1 ∞  e ( ) e ( )   − lim  ∫  −  dk  ,  →0 π  0  ik ( r − i ) ik ( r + i )   (2.9) or i 1∞ 1 1 PV ∫ PV e −ikr dk = PV , π r∞ k r (2.10) where we used (see Ref. [7]) ∞ 1 ∫ PV k e ∞ − ikx dk = π Sgn ( x ) , i (2.11) together with Sgn ( r ) = 1 , where Sgn ( x ) is the function sign of x. 3. Materials Needed from Fetter and Walecka’s Book 3.1. Self Energies The energy that a particle gains as the result of environment-modifications that it itself generates is called a self-energy Σ . This quantity denotes the contribution to the particle’s effective mass due to interactions particle-surrounding medium (SM). Consider the particular (and common) condensed matter scenario: electrons moving in a material. Σ represents there the potential felt by a given electron due to the SM’s interactions with it. Given that electrons repel each other, a moving electron does polarize the electrons in its vicinity, This, in turn, changes the potential of the moving electron fields. Such effects necessarily involve self-energy. 3.2. Fermion Dressed Propagators The dressed propagator is defined to be the two-point function to all orders of the perturbation expansion. It changes the bare mass to the physical mass. We DOI: 10.4236/jmp.2020.116056 922 Journal of Modern Physics M. C. Rocca, A. Plastino will use this notion here. For an accessible discussion of the concept we recommend the book [8]. In Fetter and Walecka’s (FW) [6] one, this idea is comprehensively discussed for a fermion’s NR QFT. In the case of free fermions, FW defined the following (current) propagator 0 iGαβ ( x, t; x ′, t ′ ) = 0 T ψ α ( x, t )ψ β+ ( x ′, t ′ ) 0 . (3.1) One has 0 iGαβ ( x, t; x ′, t ′ ) = δαβ ( 2π ) 3 ∫e i k ⋅( x − x ′ ) − ω k ( t − t ′ ) e Θ ( t − t ′ ) Θ ( k − k F ) − Θ ( t ′ − t ) Θ ( k F − k )  d 3 k . (3.2) Θ is the Heaviside’s step function. We appeal now to the very well known relation Θ ( t − t ′ ) =− 1 ∞e ( ) ∫ ω + i0 dω , 2πi −∞ − iω t − t ′ (3.3) and find 0 iGαβ ( x, t; x ′, t ′ ) δαβ = ( 2π ) ∞ 3 ∫∫e i k ⋅( x − x ′ ) − ω k ( t − t ′ ) −∞ e  Θ ( k − kF ) Θ ( kF − k )  3 −   d kdω.  ω − ωk + i 0 ω − ωk − i 0  (3.4) Thus, the pertinent expression in momentum space reads  Θ ( k − kF ) Θ ( kF − k )  Gˆ F0αβ ( k , ω ) δαβ  = + ,  ω − ωk + i 0 ω − ωk − i 0  (3.5) with 1 1 = PV  iπδ (ω − ωk ) , ω − ωk ± i 0 ω − ωk (3.6) 2 where k = k and ωk = k 2m We already stated above that PV signifies ‘‘principal value of a function’’. The system’s interaction’s Hamiltonian is defined by a two-body VF potential such that VF ( x1 − x2 = ) VF ( x1 − x2 ) 1 (1) 1 ( 2 ) , (3.7) where 1 is the unity matrix. The dressed propagator here verifies Gˆ Fαβ = δαβ Gˆ F , (3.8) so that the dressed propagator becomes diagonal. Then, ( Gˆ F0 ( k , ω ) ≡ Gˆ F0 ( k ) ) Gˆ F ( k ) = Gˆ F0 ( k ) + Gˆ F0 ( k ) Σ F ( k ) Gˆ F0 ( k ) , (3.9) with Σ F ( k ) the self-energy. We can pass now to its perturbative expansion at first order ) Σ(F ) ( k ) ≡ Σ(= (k ) 1 1 n ˆ 1 V (0) − VˆF ( k − k ′ ) Θ ( k F − k ′ ) d 3 k ′, ∫ 3  ( 2π )  (3.10) with n = N V and DOI: 10.4236/jmp.2020.116056 923 Journal of Modern Physics M. C. Rocca, A. Plastino VˆF ( k ) = ∫ VF ( x ) e − ik ⋅ x d 3 x. (3.11) Consequently (up to first order), 1 1 Gˆ F( ) ( k ) = Gˆ F0 ( k ) + Gˆ F0 ( k ) Σ(F ) ( k ) Gˆ F0 ( k ) . (3.12) 3.3. Bosons’ Dressed Propagators from FW’s Book For free bosons FW introduce the propagator in momentum space as iG 0 ( x , t ; x ′, t ′ ) = 0 T φ ( x , t ) φ + ( x ′, t ′ )  0 . (3.13) It reads Gˆ B0 ( k ) = 1 , k 0 − ωk + i 0 (3.14) 2 with ωk = k 2m . One has then 4 Gˆ B ( k ) = − ( 2π ) n0 iδ ( k0 , k ) + Gˆ B′ ( k ) , (3.15) where the primed part refers to the noncondensate ( n0 = N 0 V ) 4 1 Gˆ B ( k ) = − ( 2π ) n0 iδ ( k0 , k ) + Gˆ B0 ( k ) + Gˆ B′( ) ( k ) , 1 = Gˆ B′( ) ( k ) (3.16) n0 ˆ 0 GB ( k ) VˆB ( 0 ) + VˆB ( k )  Gˆ B0 ( k ) , h (3.17) and VˆB ( k ) = ∫ VB ( x ) e − ik ⋅ x d 3 x. (3.18) 4. Non-Relativistic QFT of Newton’s Gravity 4.1. Fermions We wish to calculate Σ(1) for the potential − VF ( r ) = − Gm 2 . r Gm 2 . r (4.1) One has VˆF ( k ) = ∫ VF ( x ) eik ⋅ x d 3 x, (4.2) 4πGm VˆF ( k ) = − , k2 (4.3) VˆF ( 0 ) = 0. (4.4) and then 2 with Starting here, a father lengthy manipulation leads to −4πGm 2 ∫ Θ ( kF − k ′) k − k′ 2 2 k 2 − k 2  kF + k  −8π2 Gm 2 F d3 k ′ = ln   , 2k  kF − k  (4.5) so that the self energy reads DOI: 10.4236/jmp.2020.116056 924 Journal of Modern Physics M. C. Rocca, A. Plastino 2 Gm 2 k 2 − k 2  k F + k  1 Σ( ) ( k ) = F ln   . 2k π  kF − k  (4.6) Accordingly, one writes for the dressed propagator 2 2 Gm 2 k F2 − k 2  k F + k   ˆ 0 (1) ln  Gˆ = Gˆ F0 ( k ) +  GF ( k )  , F (k ) 2k π  kF − k  (4.7)  Θ ( k − kF ) Θ ( kF − k )  0 = Gˆ F0 ( k ) Gˆ= +  . F ( k,ω )  ω − ωk + i 0 ω − ωk − i 0  (4.8) noting that We recall at this stage that, in Ref. [9], it was been proved that ( −1) 1 ( m) δ ( x) PV = xn n m! ( m + n )! 2 ⋅δ ( m+ n) ( x). (4.9) Then, using the result PV 1 1 1 PV m = PV ( n + m ) , xn x x (4.10) we reach 1 1 ω − ωk − i 0 ω − ωk − i 0 = 1 ( ω − ωk − i 0 ) 2 (4.11) , so that  Θ ( k − kF ) 2 Θ ( kF − k )  Gˆ F0 ( k , ω )   , = + 2 2    (ω − ωk + i 0 ) (ω − ωk − i 0 )  (4.12) If V → ∞ , k F → ∞ , n finite, we find 1 ∫ k − k ′ 2 d k ′ = 0, (4.13) Σ(F ) ( k )  0, (4.14) 1 Gˆ F( ) ( k )  Gˆ F0 ( k ) . (4.15) 3 so that 1 and thus 4.2. Bosons’ Potential VB ( r ) We calculate now the dressed propagator for VB ( r ) = − Gm 2 . r (4.16) Since 4πGm VˆB ( k ) = − , k2 2 (4.17) one has DOI: 10.4236/jmp.2020.116056 925 Journal of Modern Physics M. C. Rocca, A. Plastino VˆB ( 0 ) = 0. (4.18) For this result, we have used the relation of [7] concerning the regularization of integrals that depend upon a power of x. Thus, for the dressed propagator we find, up to first order 2 n 4πGm 2 ˆ 0 1 GB ( k )  . Gˆ B′( ) ( k ) = − 0 2   h k (4.19) We must proceed from here as we did for the fermion case to obtain 2 1 Gˆ B0 ( k )  = , 2   ( k 0 − ωk + i 0 ) (4.20) and we obtain for the dressed propagator the relation 2 n 4πGm 2 ˆ 0 GB ( k )  . Gˆ B ( k ) = −in0 2π4δ ( k0 , k ) + Gˆ B0 ( k ) − 0 2   h k (4.21) 5. Conclusions We have here applied a recently developed non relativistic quantization methodology [2] [9] [10] [11] [12] to Newton’s gravitation potential. • We emphasize that our methodology successfully tackles all renormalization issues. We made full use ultra-hyperfunctions’ theory, in particular the results reported in [2]. • With such tools we have been able to construct a non-relativistic quantum field theory (NR QFT) of Newton’s gravitation (NG). • This was done for pairs of fermions or bosons that interact between themselves via NG. • Our manipulations were based on the results of the classical book [6]. • As special examples, we have obtained the dressed propagators for both types of particles, up to first order in perturbation theory, and also the fermions’ self-energy. • The examples indicate that we have indeed constructed, both for fermions and bosons, a viable non-relativistic quantum field theory of gravitation. • Remark that we were here concerned only with Newton’s gravitation. Conflicts of Interest The authors declare no conflicts of interest regarding the publication of this paper. References DOI: 10.4236/jmp.2020.116056 [1] Plastino, A. and Rocca, M.C. (2020) Annals of Physics, 412, Article ID: 168013. https://doi.org/10.1016/j.aop.2019.168013 [2] Plastino, A. and Rocca, M.C. (2018) Journal of Physics Communications, 2, Article No. 11. https://doi.org/10.1088/2399-6528/aaf186 [3] Plastino, A. and Rocca, M.C. (2020) Journal of Modern Physics, 11, 565-580. https://doi.org/10.4236/jmp.2020.114037 926 Journal of Modern Physics M. C. Rocca, A. Plastino [4] Plastino, A. and Rocca, M.C. (2020) Journal of High Energy Physics, Gravitation and Cosmology, 6 298-311. https://doi.org/10.4236/jhepgc.2020.62023 [5] Plastino, A. and Rocca, M.C. (2020) Journal of Physics Communications, 4, Article No. 3. https://doi.org/10.1088/2399-6528/ab8178 [6] Fetter, A.L. and Walecka, J.D. (1971) Quantum Theory of Many-Particle Systems. McGraw-Hill Book Company, New York. [7] Guelfand, I.M. and Shilov, G.E. (1972) Les Distributions. Vol. 1, Dunod, Paris. [8] Matttuck, R.D. (1996) A Guide to Feynman Diagrams in the Many Body Problem. 2nd Edition, McGraw Hill, NY. [9] Bollini, C.G., Escobar, T. and Rocca, M.C. (1999) International Journal of Theoretical Physics, 38, 2315-2332. https://doi.org/10.1023/A:1026623718239 [10] Bollini, C.G. and Rocca, M.C. (2004) International Journal of Theoretical Physics, 43, 59-76. https://doi.org/10.1023/B:IJTP.0000028850.35090.24 [11] Bollini, C.G. and Rocca, M.C. (2004) International Journal of Theoretical Physics, 43, 1019-1051. https://doi.org/10.1023/B:IJTP.0000048599.21501.93 [12] Bollini, C.G., Marchiano, P. and Rocca, M.C. (2007) International Journal of Theoretical Physics, 46, 3030-3059. https://doi.org/10.1007/s10773-007-9418-y DOI: 10.4236/jmp.2020.116056 927 Journal of Modern Physics 

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An Introduction to Audit Studies in the Social Sciences S. Michael Gaddis mgaddis@soc.ucla.edu Assistant Professor Department of Sociology University of California – Los Angeles January 2018 Pre-Publication Draft: Please see footnote for citation. 1 Keywords: audit studies, correspondence audits, discrimination, field experiments Abstract: An audit study is a specific type of field experiment primarily used to test for discriminatory behavior when survey and interview questions induce social desirability bas. In this chapter, I first review the language and definitions related to audit studies and encourage adoption of a common language. I then discuss why researchers use the audit method as well as when researchers can and should use this method. Next, I give an overview of the history of audit studies, focusing on major developments and changes in the overall body of work. Finally, I discuss the limitations of correspondence audits and provide some thoughts on future directions. Acknowledgments: Many scholars have played important roles in sharpening my thoughts on this method. I cannot name them all here but I want to express my thanks to Devah Pager, Bill Carbonaro, Joanna Lahey, and David Pedulla for support in helping this volume come together. Additionally, I would like to thank fellow panelists and audience members at sessions on audits at the 2014 annual meeting of the Association for Public Policy Analysis and Management in Albuquerque, NM and the 2015 annual meeting of the American Sociological Association in Chicago, IL. 1 Gaddis, S. Michael. 2018. “An Introduction to Audit Studies in the Social Sciences.” In Audit Studies: Behind the Scenes with Theory, Method, and Nuance, edited by S. M. Gaddis, p3-p44. Cham, Switzerland: Springer International Publishing. INTRODUCTION Since the 1960s, researchers have had a methodological tool at their disposal unlike any other: the audit study. 2 The audit study is a specific type of field experiment that permits researchers to examine difficult to detect behavior, such as racial and gender discrimination, and decision-making in real-world scenarios. Audit studies allow researchers to make strong causal claims and explore questions that are often difficult or impossible to answer with observational data. This type of field experiment has exploded in popularity in recent years, particularly to examine different types of discrimination, due to the rise of online applications for housing and employment and easy access to decision makers across many contexts via email. However, the learning curve for designing and implementing these experiments can be quite steep, despite appearing to be a simple and quick method for examining discrimination. Thus, we have written this book to help scholars design, conduct, and analyze their own audits. This book draws upon the knowledge of a variety of social scientists and other experts who combined have implemented dozens of in-person and correspondence audits to examine a variety of research questions. These experienced scholars share insights from both their successes and failures and invite you, the reader, “behind the scenes” to examine how you might construct your own audit study and improve upon this method in the future. We write this book with a wide audience in mind and hope that you will find this book useful whether you have already fielded your own audit study, are just thinking about how you might design an audit study, or just want to learn more about the method to better understand research using audits. 2 These types of studies are known by a variety of names, often depending on the decade of publication, the context and method used for testing, discipline, or country. Audits are also sometimes referred to as correspondence tests or situation tests. For now, I refer to all this research as “audit studies.” Later in this chapter, I define and clarify these terms. In this introductory chapter, I approach the subject as one might with a lay audience. However, even experienced researchers with in-depth knowledge of the audit method should find this chapter useful. I mostly focus on the aspects of audit studies related to research rather than those related to activism or law and policy. 3 I begin this chapter with the basics – a discussion of the language and definitions related to audit studies. Significant differences in language persist between studies, researchers, and disciplines, and I hope that this section will help readers understand these differences as well as encourage researchers to adopt a common language. Next, I give a succinct overview of why researchers began using audits to examine discrimination. The audit method is a powerful tool to answer certain types of questions and I attempt to outline when researchers can and should use this method. I then give an overview of the history of audit studies. Although others have written superb reviews of this body of literature in the past (Baert this volume; Oh and Yinger 2015; Riach and Rich 2002; Zschirnt and Ruedin 2016), I focus on the forest rather than the trees in this section and provide a narrative of the arc of audit studies over time. 4 Finally, I close this chapter with a succinct discussion of the limitations of correspondence audits and thoughts on how we might improve this method. This is a not a comprehensive section, but I do think it complements the closing chapter of this book (Pedulla this volume). Readers looking for additional information on audit studies should consult two resources. First, we have created a website – www.auditstudies.com – to go along with the release of this volume. There you will find a comprehensive database of audits as well as additional 3 For an excellent chapter on the connections to activism, see Cherry and Bendick (this volume) and for an excellent, although a bit outdated, chapter on the links between audits and law and policy, see Fix, Galster, and Struyk 1993. 4 Some of the work in this section stems from and expands upon work I did to examine the signals of race conveyed by names in correspondence audits (Gaddis 2017a, 2017b, 2017c, forthcoming). information. Second, at the end of this chapter I provide a brief recommended reading section of important comprehensive works, reviews, and other methods-based articles and books. Beyond this introductory chapter, several accomplished scholars present their expert knowledge about audit studies. In the first section – The Theory Behind and History of Audit Studies – the authors cover a wide range of history, explain why we should conduct audit studies, examine the connections between audit studies and activism, and outline what researchers have uncovered about labor market processes using audit studies in the past decade. In the second section – The Method of Audit Studies: Design, Implementation, and Analysis – the experts provide guidance on designing your own audit study, discuss the challenges and best practices regarding email, review extensive issues of validity, and consider the technical setup of matching procedures. In the final section – Nuance in Audit Studies: Context, Mechanisms, and the Future – the authors focus on more nuanced aspects of audit studies and address limitations and challenges, examine the use of context to explore mechanisms, and consider the value of variation. I return to a brief discussion of the rest of this book at the end of this chapter. THE BASICS OF AUDIT STUDIES: LANGUAGE AND DEFINITIONS Field experiments encompass a wide range of studies and ideas and describe the highest level of the hierarchy I focus on here. Audit studies are one type of field experiment. At their core, field experiments in the social sciences attempt to mimic the experiments of the natural sciences by implementing a randomized research design in a field setting (as opposed to a lab or survey setting). Although many may think of psychology as the disciplinary home to social science experiments, researchers in economics, political science, and sociology have ramped up the quantity and quality of field experiments conducted in these disciplines over the past few decades. Although not the only reason for the increase in field experiments across these disciplines, audit studies do represent a major part of the heightened activity. Audit studies generally refer to a specific type of field experiment in which a researcher randomizes one or more characteristics about individuals (real or hypothetical) and sends these individuals out into the field to test the effect of those characteristics on some outcome. Historically, audit studies have focused on race and ethnicity (Daniel 1968; Bertrand and Mullainathan 2004; Wienk et al. 1979) and gender (Ayres and Siegelman 1995; Levinson 1975; Neumark, Bank, and Van Nort 1996). In recent years, researchers have expanded the manipulated characteristics to include age (Ahmed, Andersson, and Hammarstedt 2012; Bendick, Jackson, and Romero 1997; Farber, Silverman, and von Wachter 2017; Lahey 2008; Neumark, Burn, and Button 2016; Riach 2015; Riach and Rich 2010), criminal record (Baert and Verhofstadt 2015; Evans 2016; Evans and Porter 2015; Furst and Evans 2016; Pager 2003), disability (Ameri et al. forthcoming; Baert 2014a; Ravaud, Madiot, and Ville 1992; Turner et al. 2005; Verhaeghe, Van der Bracht, and Van de Putte 2016), educational credentials (Carbonaro and Schwarz this volume; Darolia et al. 2015; Deming et al. 2016; Deterding and Pedulla 2016; Gaddis 2015, 2017d; Jackson 2009), immigrant assimilation or generational status (Gell-Redman et al. 2017; Ghoshal and Gaddis 2015; Hanson and Santas 2014), mental health (Baert et al. 2016a), military service (Baert and Balcaen 2013; Figinski 2017; Kleykamp 2009), parental status (Bygren, Erlandsson, and Gähler 2017; Correll, Benard, and Paik 2007; Petit 2007), physical appearance (Bóo, Rossi, and Urzúa 2013; Galarza and Yamada 2014; Maurer-Fazio and Lei 2015; Patacchini, Ragusa, and Zenou 2015; Ruffle and Shtudiner 2015; Stone and Wright 2013), religious affiliation (Adida, Laitin, and Valfort 2010; Pierné 2013; Wallace, Wright, and Hyde 2014; Wright et al. 2013), sexual orientation (Ahmed, Andersson, and Hammarstedt 2013; Baert 2014b; Bailey, Wallace, and Wright 2013; Drydakis 2009, 2011a, 2014; Mishel 2016; Tilcsik 2011; Weichselbaumer 2015), social class (Heylen and Van den Broeck 2016; Rivera and Tilcsik 2016), and spells of unemployment and part-time employment (Birkelund, Heggebø, and Rogstad 2017; Eriksson and Rooth 2014; Kroft, Lange, and Notowidigdo 2013; Pedulla 2016), among other characteristics (Baert and Omey 2015; Drydakis 2010; Kugelmass 2016; Tunstall et al. 2014; Weichselbaumer 2016). The “individuals” sent into the field may be actual people in an in-person audit or simply applicants or emails from hypothetical people in correspondence audits (more below). The outcomes may be an offer to interview for a job (Bertrand and Mullatinathan 2004; Darolia et al. 2015; Deming et al. 2016; Gaddis 2015), a job offer (Bendick, Jackson, and Reinoso 1994; Bendick, Rodriguez, and Jayaraman 2010; Pager, Western, and Bonikowski 2009; Turner, Fix, and Struyk 1991), the order in which applicants are contacted (Duguet et al. 2015), a response to a housing inquiry (Ahmed and Hammarstedt 2008; Bengtsson, Iverman, and Hinnerich 2012; Carlsson and Eriksson 2014; Carpusor and Loges 2006; Ewens, Tomlin, and Wang 2014; Feldman and Weseley 2013; Hogan and Berry 2011; Van der Bracht, Coenen, and Van de Putte 2015), the types of housing shown (Galster 1990a; Turner et al. 2002, 2013), information about the availability of a house for purchase or rent (Galster 1990b, Turner et al. 2002, 2013; Yinger 1986), an offer of different housing than requested or racial steering (Galster and Godfrey 2005; Turner, Mikelsons and Edwards 1990), a response to a mortgage application or request for information (Hanson et al. 2016; Smith and Cloud 1996; Smith and DeLair 1999), a response to a roommate request (Gaddis and Ghoshal 2015, 2017; Ghoshal and Gaddis 2015), an offer to schedule a doctor’s appointment (Kugelmass 2016; Sharma, Mitra, and Stano 2015), a response from a politician or other public official (Broockman 2013; Butler and Broockman 2011; Chen, Pan, and Xu 2016; Distelhorst and Hou 2014; Einstein and Glick 2017; Hemker and Rink forthcoming; Janusz and Lajevardi 2016; McClendon 2016; Mendez and Grose 2014; White, Nathan, and Faller 2015), a response from a professor (Milkman, Akinola, and Chugh 2012, 2015; Zhao and Biernat 2017), the price paid or bargained for economic transactions for goods (Anagol, Cole, and Sarkar 2017; Ayres 1991; Ayres and Siegelman 1995; Besbris et al. 2015; Doleac and Stein 2013), or a number of other outcomes (Allred et al. 2017; Edelman, Luca, and Svirsky 2017; Giulietti, Tonin, and Vlassopoulos 2015; Ridley, Bayton, and Outtz 1989; Wallace et al. 2012; Wissoker, Zimmermann, and Galster 1998; Wright et al. 2015). Two main variations of audits exist: in-person audits and correspondence audits. Inperson audits rely on trained assistants to conduct the experiment. Early audit studies almost exclusively referred to the research subjects posing as legitimate applicants for employment or housing as testers or auditors. This is due, in part, to the fact that the language for such research was adopted from early testing for legal violations for enforcement rather than research purposes (see Boggs, Sellers, and Bendick 1993 and Fix and Turner 1999 for an in-depth discussion of differences between paired testing for enforcement purposes versus research). However, as correspondence audits overtook in-person audits as the norm and real individuals posing as subjects were not required, researchers shifted their language to refer to applicants, candidates, constituents, prospective tenants, etc. In other words, the language should match what the audit context dictates. Although the language identifying testers, auditors, or applicants may vary due to the nature of the study, we recommend that researchers adopt a common language of “inperson audits” to identify field cases using live human beings and “correspondence audits” to identify online, telephone, or by mail audits using hypothetical individuals or recorded messages in the case of some audits by telephone. Although most audit studies include paired (or sometimes triplet) testing with comparisons of two (or three) testers or applicants, not all do (for example, see Hipes et al. 2016; Lauster and Easterbrook 2011; Rivera and Tilcsik 2016). Paired testing, also referred to as matched testing, is a design in which the subject or organization being audited (e.g., employer, real estate agent, etc.) receives applications or emails from two or more of testers with different characteristics. Conversely, non-paired testing is a design in which the subject or organization being audited only ever receives a single tester application or email. For example, a paired test design might send both a black couple and a white couple to each real estate agent’s office in the sample whereas a non-paired test design would send only one of the two couples (randomly) to each real estate agent’s office in the sample. There can be statistical advantages to paired testing, however, in some cases it may be necessary to implement a non-paired test design to reduce suspicion and avoid experiment discovery (Vuolo, Uggen, and Lageson 2016, this volume; Weichselbaumer 2015, 2016). THE NEED FOR AUDIT STUDIES 5 Not coincidentally, the rise of audit studies by researchers corresponds with the public policy of the civil rights era aimed to stop racial discrimination and reduce, if not eliminate, racial inequality. Prior to the 1960s, racial discrimination in the United States occurred openly in public, was relatively common, had minimal stigma attached to it, was shaped by open prejudicial attitudes and beliefs, and arguably was informed by a conscious or active racial prejudice. Individual employers, real estate agents, and landlords could discriminate with impunity and often made public their beliefs and actions. In the United States, the Civil Rights 5 In this section, I discuss audits studies from the perspective of racial discrimination. However, the need for and use of audits is similar across other types of discrimination as well as some non-discrimination-based domains of inquiry. Act of 1964 intended to change these behaviors, if not beliefs and attitudes, by outlawing discrimination on the basis of race, color, religion, sex, or national origin. The Equal Employment Opportunity Commission (EEOC) gained the ability to litigate discrimination cases following the passage of the Equal Employment Opportunity Act in 1972. Title VII of the Civil Rights Act of 1964 finally could be enforced. However, we can imagine and, indeed do live in, a world where the Civil Rights Act may have changed the act of discrimination without changing the amount of discrimination, intentions behind discrimination, or desire to discriminate. Although not a sharp change overnight, discrimination of all types has changed in response to the Civil Rights Act. Modern discrimination has become more covert, uncommon, and stigmatized, while being shaped by private prejudicial attitudes and beliefs, and, perhaps, informed by an unconscious or latent racial prejudice. Individuals may fear litigation for engaging in discrimination or have a social desirability bias to not acknowledge discriminatory actions. This makes it difficult for researchers to document and examine discrimination. Thus, two traditional methods of social science inquiry are difficult, if not impossible, to employ to examine discrimination in the post-civil rights era. First, pointed interviews and survey questions asking perpetrators about racial discrimination are unlikely to elicit truthful responses. To my knowledge, the most recent research project to successfully elicit clearly truthful responses from employers about engaging in racial discrimination occurred in the late 1980s (Kirschenman and Neckerman 1991). Moreover, surveys and interviews do not document actions, but rather self-reported beliefs, attitudes, recollections of past actions, or predictions of future actions. Due to respondents’ fear and social desirability bias, and the sometimes unconscious nature of racial prejudice, direct questions about discrimination through interviews and surveys exhibit low construct validity. Second, statistical analyses using secondary data that do not have explicit questions about discrimination also fail to adequately capture discrimination. To understand the difficulty of this process, let’s first consider a definition of discrimination. In a 2004 book stemming from the Committee on National Statistics’ Panel on Methods for Assessing Discrimination, panelists defined racial discrimination as “differential treatment on the basis of race that disadvantages a racial group” (Blank et al. 2004: 39). Although researchers can document the second (race) and third parts (disadvantage) of the definition with secondary data, directly capturing the first part (differential treatment) is impossible. Thus, secondary data analysis must use indirect residual attribution to suggest that, after including a litany of control variables that affect the dependent variable of interest on which blacks and whites differ, any remaining coefficient for race represents discrimination (Blank et al. 2004; Lucas 2008; Neumark forthcoming). However, this method is unlikely to correctly attribute the true amount of racial discrimination (Quillian 2006), due to omitted variable bias, among other issues (Altonji and Blank 1999; Blank et al. 2004; Farkas and Vicknair 1996; Lucas 2008). Researchers developed the audit method as a means of catching individuals and organizations in the act of discrimination. Generally, experiments can be done when a presumed cause is manipulable and should be done when it is otherwise difficult to prove nonspuriousness. Many, if not all, types of discrimination are great candidates for examination through experimental means because the presumed cause often is manipulable in many contexts and, as discussed earlier, traditional methods of social science inquiry have been unable to directly document discrimination or rule out a spurious relationship. If we consider the previously stated definition of racial discrimination – “differential treatment on the basis of race that disadvantages a racial group” (Blank et al. 2004: 39) – we see that audit studies manipulate the second part (race) to directly capture the first part (differential treatment) of the definition. Thus, by carefully controlling and counterbalancing all other variables in the experimental process, audit studies provide strong causal evidence of discrimination. A HISTORY OF AUDIT STUDIES The Early Years: The First In-Person and Correspondence Audits In-person audits began in the 1940s and 1950s by means of activists and private organizations with some assistance from academic researchers. One of the earliest media mentions of audits occurred in the New York Times in 1956 (Rowland). In Chapter 2, Frances Cherry and Marc Bendick Jr. (this volume) do an excellent job of covering some of this early work, so I leave discussion of that part of the history of audit studies to them. The earliest known published audit study of significant scope and scale was conducted in England in the late 1960s. With the Race Relations Acts of 1965, Parliament passed the first legislation addressing racial discrimination in the United Kingdom in public domains. The following year, the U.K. Parliament created the Race Relations Board, which was tasked with reviewing complaints falling under the Race Relations Act. However, the Race Relations Act did not cover employment and housing discrimination until 1968, so in tandem with the National Committee for Commonwealth Immigrants, the Race Relations Board commissioned a study on racial discrimination in employment, housing, and other contexts. Along with surveys and interviews, the study implemented the audit method to extensively examine discrimination (Daniel 1968). Described as “situation tests,” the audits were born when Daniel and the research team had doubts over whether surveys and interviews would give them an accurate portrayal of the state of discrimination. Moreover, the team was unsure if the “findings would appear conclusive to those people who are strongly passionate or committed about the subject on one side or the other” (1968: 20). That doubt led them “not to depend entirely on what people told us in interviews, but to put the matter to the test in a way that would provide objective evidence” (ibid). These tests were conducted with triplets of candidates – usually white English, white immigrant, and black applicants – in the domains of housing (both rental and purchase), employment, and other services. The tests consistently uncovered discrimination against blacks and immigrants. At the time, this commissioned study of racial discrimination was monumentally important. Along with the hard work of researcher William Wentworth Daniel, results from this study led to the revised Race Relations Act of 1968 outlawing racial discrimination in employment and housing (Smith 2015). However, this study often has been overlooked or forgotten by academics; at the time of this writing, Google Scholar reports that the resulting book by Daniel (1968) has garnered fewer than 500 citations in nearly fifty years. Still, Racial Discrimination in England’s use of the audit method in government-sponsored research marks the beginning of a series of high profile in-person audits conducted to examine racial discrimination. Just a few years later, in 1969, the first-ever correspondence audit was conducted in the United Kingdom. Published by two researchers from the non-profit institute Social and Community Planning Research, this study sought to examine racial discrimination among employers looking to hire white-collar workers (Jowell and Prescott-Clarke 1970). The authors chose to conduct a correspondence audit through the mail because “postal applications were possible and, in many cases, necessary” to apply for employment (1970: 399). The authors matched British-born whites with four different immigrant groups to test for racial discrimination across an ambitious-for-the-time 128 job postings (256 total applicants) and noted the importance of both realism in the application and controlling for all differences between candidates including aspects such as handwriting. Again, although this study has collected few citations in nearly fifty years (fewer than 150 at the time of this writing), it remains an incredibly important entry in the annals of the audit method because it introduced the world to correspondence audits. The First Wave: The Early 1970s through the Mid 1980s In the United States, a number of non-academic-based audits followed the two UK studies. Private fair housing audits rose to prominence in the late 1960s and 1970s in the United States following passage of the Civil Rights Act of 1968 (also known as the Fair Housing Act), which provided federal enforcement of anti-discrimination housing law through an office of the U.S. Department of Housing and Urban Development (HUD). These audits were often conducted in partnership with academic researchers (often local) and often focused on one major city, such as Akron, Ohio (Saltman 1975), Chicago (as reported in Cohen and Taylor 2000), Detroit (Pearce 1979), Los Angeles (Johnson, Porter, and Mateljan 1971), and New York (as reported in Purnell 2013). Additionally, organizations often produced method-based manuals and guides for the practice of auditing (Kovar 1974; Leadership Council for Metropolitan Open Communities 1975; Murphy 1972). However, the largest, and arguably most important, audit on housing discrimination during this era, the Housing Market Practices Survey (HMPS), occurred in 1977 (Wienk et al. 1979). This first large-scale housing audit was commissioned by HUD to test for discrimination against blacks in both the sale and rental housing markets. HUD paired with local fair housing organizations and other organizations to recruit and train testers to conduct the in-person audits. This research included 3,264 audits across forty metro areas, with a plurality of the audits occurring in five metro areas. The HMPS found discrimination against blacks in reported housing availability, treatment by real estate agents, reported terms and conditions, and the types and levels of information requested by real estate agents. This research was critically important in leading the way for future audits, including three additional national housing audits commissioned by HUD (Turner and James 2015; Turner et al. 2002, 2013; Turner, Struyk, and Yinger 1991; Yinger 1991, 1993), several smaller local audits (see below), and the Urban Institute employment audits a decade later (Cross et al. 1990; Mincy 1993; Turner, Fix, and Struyk 1991). Arguably, four aspects of the HMPS were important in shaping future audits. First, the HMPS showed that large-scale audits for discrimination in the United States were possible. Second, this research essentially gave auditing a gold seal of approval from an arm of the federal government (for more details on audits and the courts, see Boggs, Sellers, and Bendick 1993; Fix, Galster, and Struyk 1993; and Pager 2007a). Third, it was the first research to show the extent to which racial discrimination was widespread. Finally, the HMPS showed creativity in expanding the outcomes examined by audits. Other one-off in-person and correspondence audits conducted during the 1970s and early 1980s examined housing and employment discrimination in the United Kingdom (McIntosh and Smith 1974), housing discrimination in France (Bovenkerk et al. 1979) and the United States (Feins and Bratt 1983; Galster and Constantine 1991 6; Hansen and James 1987; James, McCummings, and Tynan 1984; Newburger 1984; Roychoudhury and Goodman 1992, 1996 7), and employment discrimination in the United States (Hitt, Zikmund, and Pickens 1982; Jolson 1974; Levinson 1975; McIntyre, Moberg, and Posner 1980; Newman 1978), Canada (Adam 1981; Henry and Ginzberg 1985), Australia (Riach and Rich 1987, 1991), and England (Brown and Gay 1985; Firth 1981; Hubbuck and Carter 1980). George Galster (1990a, 1990b) reviewed several fair housing audits conducted in the 1980s that were mostly unpublished and analyzed data from seventy-one separate audits. During this period, researchers also began to expand the domains in which they investigated discrimination. As early as 1985, Galster and Constantine (1991) investigated housing discrimination based on parental and relationship status among women. Ayres (1991 and Ayres and Siegelman 1995) examined racial and gender discrimination in bargaining for new car prices, while Ridley Bayton and Outtz (1989) examined racial discrimination in hailing a taxi. Other research from this period examined discrimination based on disability (Fry 1986; Graham, Jordan, and Lamb 1990; Ravaud, Madiot, and Ville 1992). The first wave of audits conducted in the 1970s and 1980s filled in a number of gaps in our knowledge about the extent and geography of discrimination, conditions under which discrimination occurred, and variations in outcomes that were affected by discrimination, particularly in housing and, to some degree, employment. The Second Wave: The Late 1980s through the Late 1990s Beginning with the last part of the 1980s and continuing throughout the 1990s, a second wave of audits was ushered in with the second iteration of the HUD housing audit (Turner, 6 7 Conducted in 1985 Conducted throughout the 1980s. Micklensons, and Edwards 1991; Yinger 1991, 1995) and a series of large-scale employment audits conducted by the Urban Institute (Cross et al. 1990; Mincy 1993; Turner, Fix, and Struyk 1991), in part, aided by guidelines for adapting housing audits to hiring situations (Bendick 1989). The HUD housing audit in 1989, known as the Housing Discrimination Study (HDS) 1989, was conducted in partnership with the Urban Institute. The HDS 1989 varied from and improved on the HMPS in 1977 in many ways. First, the former included Hispanic testers paired with whites for some audits to examine discrimination against Hispanics as well (Ondrich, Stricker, and Yinger 1998; Page 1995), something that was only done in an extension of the HMPS and only in Dallas (Hakken 1979). Second, in the HDS 1989 auditors focused on specific advertised housing units, whereas in the HMPS auditors approached agents about more general housing options fitting certain criteria. Thus, the HDS 1989 could more accurately examine racial steering. Third, the HDS 1989 examined fewer metro areas (25 instead of 40), but conducted more audits (3,800 instead of 3,264). Overall, the HDS 1989 replicated the general finding of the HMPS that housing discrimination against blacks was prevalent and widespread. However, there was no strong evidence suggesting that discrimination increased or decreased between the two data collection periods (Elmi and Mikelsons 1991). The first of the Urban Institute employment audits was conducted in Chicago and San Diego in 1989 and examined discrimination against Hispanics (Cross et al. 1990). Researchers sampled newspaper advertisements and matched pairs successfully applied to almost 300 entrylevel jobs in the two cities. The study found that Hispanics faced discrimination at both the application and interview phases, which lead to fewer interviews and fewer job offers when compared with their white counterparts. In 1990, the Urban Institute conducted a similar employment audit in Chicago and Washington, D.C. to examine discrimination against African Americans (Turner, Fix, and Struyk 1991). Matched pairs successfully completed nearly 450 audits in the two cities. The study found that employers discriminated against blacks in accepting their applications, inviting them to interview, and offering them a job. Black applicants were also more likely to be steered toward lower quality jobs rather than the advertised position to which they responded. Additionally, whites were treated more favorably in a number of respects, including waiting time, length of interview, and positive comments. The Urban Institute studies were the first large-scale true employment audits conducted in the U.S. Researchers and staff went to great lengths to make the study as methodologically sound as possible and paid close attention to detail in sampling, creating matched pairs, and standardizing procedures for the audits (Mincy 1993). Although these studies provided a meticulous model for subsequent researchers to follow when conducting employment audits, others have extensively critiqued the Urban Institutes studies and the in-person audit method more broadly (Heckman 1998; Heckman and Siegelman 1993). However, by moving development and knowledge of the method forward and by providing extensive guidance (along with Bendick 1989) for the numerous employment audits that followed them, the Urban Institute audits were clearly of great importance. Following the HDS 1989 and the Urban Institute employment audits, a wave of audits examining employment, housing, and other forms of discrimination occurred. Many audits were conducted in Europe through the International Labour Office (ILO) based on guidelines developed by Frank Bovenkerk (1992). Studies in the U.S. (Bendick et al. 1991; Bendick, Jackson, and Reinoso 1994; James and DelCastillo 1992; Nunes and Seligman 1999) and Europe (Arrijn, Feld, and Nayer 1998; Bovenkerk et al. 1995; de Prara et al. 1996; Esmail and Everington 1993, 1997; Goldberg, Mourinho, and Kulke 1995; Smeesters and Nayer 1998) focused on race and ethnic discrimination. Researchers conducted sex discrimination employment audits in the U.S. (Neumark, Bank, and Van Nort 1996; Nunes and Seligman 2000) and Europe (Weichselbaumer 2000), as well as age and disability-based discrimination employment audits in the U.S. (Bendick, Brown, and Wall 1999) and Europe (Graham, Jordan, and Lamb 1990; Gras et al. 1996). This period also included the return of telephone-based (Bendick, Brown, and Wall 1999; Massey and Lundy 2001; Purnell, Idsardi, and Baugh 1999) and written correspondence audits (Bendick, Jackson, and Romero 1997; Gras et al. 1996; Weichselbaumer 2000). Still, the cost-prohibitive nature of in-person audits and labor-intensive nature of correspondence audits during the 1990s meant that use of the audit method was relatively rare. The Third Wave: The Early 2000s through the Late 2000s Until the early 2000s, most audits were conducted in-person and relied on trained assistants to physically participate in the process. With housing and employment applications increasingly taking place over the internet, researchers began conducting more correspondence audits. However, some important audits in the early 2000s were still in-person, including the second iteration of HUD and the Urban Institute’s Housing Discrimination Study (HDS 2000: Bavan 2007; Ross and Turner 2005; Turner et al. 2002). Devah Pager was the first to examine the effects of a criminal record using an audit study (2003) and produced an incredibly strong body of work during this period consisting of in-person audits as well as examinations of the method (Pager 2007a, 2007b; Pager, Western, and Bonikowski 2009; Pager and Quillian 2005; Pager and Shepherd 2008; Pager, Western, and Sugie 2009). The 2000s brought about significant changes in the audit method and the importance of this era is highlighted by the fact that the two most cited audit studies of all time both occurred in the early 2000s. Devah Pager’s (2003) in-person audit study of race and criminal record in the low-wage labor market in Milwaukee has garnered nearly 1,900 citations according to Google Scholar. Marianne Bertrand and Sendhil Mullainathan’s (2004) correspondence audit study of race in labor markets in Boston and Chicago has nearly 3,000 citations at the time of this writing. Both studies have been incredibly important in shaping our understanding of racial discrimination, however, the differences between them are stark and mark a major turning point in audit studies. Bertrand and Mullainathan’s 2004 study, published in The American Economic Review, is the most influential correspondence audit study of the past two decades. In total, the authors applied to over 1,300 job advertisements, compared to Pager’s 350 jobs (2003), listed in newspapers in Boston and Chicago via fax and mail. Additionally, the authors used birth record data and a small convenience sample pretest to select names to convey race on each resume. Rather than send two applicants per job, the authors often used four resumes to examine both race and resume quality simultaneously and obtained a final sample size of 4,870. Bertrand and Mullainathan found that white applicants were about 50% more likely than black applicants to receive a callback. Moreover, black applicants benefited less than white applicants from higher resume quality. Bertrand and Mullainathan’s (2004) landmark study ushered in a new era of correspondence audits. Arguably, this study paved the way for the increase in audits that followed for at least three reasons. First, the research showed that a large-scale audit – in particular, a correspondence audit – could be undertaken by a small team of academic researchers, compared to past audits conducted by larger teams such as those at HUD and the Urban Institute. Although Bertrand and Mullainathan applied via fax and mail, the timing was ripe for the switch to applications over the internet which further expanded the possibilities of correspondence audits. Second, the study opened a dialogue about signaling race through correspondence audits. Because the authors conducted a small pretest and used a moderate number of names – 36 in total – the plurality of studies that followed used the same names to signal race (see Gaddis 2017c). 8 Although over a decade would pass before scholars began to seriously question these signals (Butler and Homola 2017; Gaddis 2017a, 2017b, 2017c, forthcoming; Weichselbaumer 2017), Bertrand and Mullainathan were the first to truly investigate them. Finally, this study showed that it was possible to successfully manipulate several characteristics simultaneously. Beyond race and gender, the authors varied other resume characteristics such as education, experience, and skills. These manipulations likely sparked ideas among researchers about mechanisms and interactions that would follow in future studies. The vast majority of the studies that followed Bertrand and Mullainathan during the 2000s were conducted via the correspondence method. A few notable exceptions are the previously mentioned studies by Devah Pager (2003; Pager, Western, and Bonikowski 2009) and three studies carried out by the International Labour Office (ILO) in Italy (Allasino et al. 2004), Sweden (Attström 2007), and France (Cediey and Foroni 2008), although the ILO studies used a mix of in-person and correspondence methods. Additionally, two in-person studies examined discrimination in market transactions: baseball card sales (List 2004) and auto repair quotes (Gneezy and List 2004). 8 Although credit should also be given to Lodder, McFarland, and White (2003) who pre-tested names in a small employment correspondence audit in Chicago before Bertrand and Mullainathan (2004). During this time, correspondence audits examining employment discrimination based on race and ethnicity expanded to cover more countries and race/ethnicities such as Albanians in Greece (Drydakis and Vlassis 2010) and Turks in Germany (Kaas and Manger 2012), and a variety of other groups in Australia (Booth, Leigh, and Varganova 2012), Canada (Oreopoulos 2011), Denmark (Hjarnø and Jensen 2008), France (Duguet et al. 2010), Great Britain (Wood et al. 2009), Ireland (McGinnity and Lunn 2011), Sweden (Bursell 2007; Carlsson 2010; Carlsson and Rooth 2007; Rooth 2010), and the U.S. (Jacquemet and Yannelis 2012; Thanasombat and Trasviña 2005; Widner and Chicoine 2011). Additionally, researchers examined employment discrimination on the basis of gender and family status in France (Petit 2007) and the U.S. (Correll, Benard, and Paik 2007), gender in England (Riach and Rich 2006a), Spain (Albert, Escot, and Fernandez-Cornejo 2011) and Sweden (Arai, Bursell, and Nekby 2016) 9, age in England (Riach and Rich 2010), France (Riach and Rich 2006b), Spain (Albert, Escot, and Fernandez-Cornejo 2011; Riach and Rich 2007), and the U.S. (Lahey 2008), sexual orientation in Austria (Weichselbaumer 2003), Greece (Drydakis 2009, 2011a) and the U.S. (Tilcsik 20011), race and criminal record in the U.S. (Galgano 2009), race and military status in the U.S. (Kleykamp 2009), educational credentials in the United Kingdom (Jackson 2009), caste in India (Siddique 2011), caste and religion in India (Banerjee et al. 2009), and physical attractiveness and obesity in Sweden (Rooth 2009). One additional study of note during this period is Philip Oreopoulos’ correspondence audit in Toronto, which included six different racial/ethnic/immigrant groups. He applied to over 3,200 job postings using 13,000 different resumes to create one of the most ambitious correspondence audits of its time. The expansion of audit research during the 2000s included housing discrimination studies as well. The HDS 2000 expanded to include Asians and Pacific Islanders as well as Native 9 Conducted in 2006 and 2007. Americans (Turner and Ross 2003a, 2003b) and examined housing discrimination on the basis of disability (Turner et al. 2005). Correspondence audits examined housing discrimination based on race and ethnicity in Canada (Hogan and Berry 2011), Greece (Drydakis 2011b), Italy (Baldini and Federici 2011), Spain (Bosch, Carnero and Farré 2010), Sweden (Ahmed, Andersson and Hammarstedt 2010; Ahmed and Hammarstedt 2008), and the United States (Carpussor and Loges 2006; Friedman, Squires, and Galvan 2010; Hanson and Hawley 2011; Hanson, Hawley, and Taylor 2011). Additional research examined housing discrimination on the basis of sexual orientation (Ahmed, Andersson and Hammarstedt 2008; Ahmed and Hammarstedt 2009). Beyond the major expansion of correspondence audits during this time, the period is marked by the beginning of researchers’ exploration of mechanisms of discrimination, intentions behind discrimination, and conditions under which discrimination occurs rather than simply documenting the existence of discrimination. At least four studies during this period attempted to uncover greater detail related to these issues. First, two studies followed up with employers after submitting them to an audit to examine bias in more detail. In one study, Devah Pager and Lincoln Quillian (2005) conducted a telephone survey to follow up with employers who had unknowingly participated months earlier in an in-person audit study. When given a vignette scenario that mimicked the audit scenario they were subjected to, employers suggested they would be much more likely to hire individuals than the callback rates suggested. In fact, the results of the vignette survey showed no differences between white and black applicants, suggesting the existence of social desirability bias. In another study, Dan-Olof Rooth (2010) administered the Implicit Association Test (IAT) to test whether discriminatory behavior in a prior correspondence audit was associated with IAT scores. He found a strong positive correlation between discrimination against Arab-Muslims 10 and IAT scores but no correlation with a separate explicit measure of bias. These results could suggest that individuals are engaging in discrimination only due to implicit bias (without having a true explicit bias) or could suggest the existence of social desirability bias. The second set of studies attempted to distinguish between statistical discrimination and taste-based discrimination. In one study, Joanna Lahey (2008) designed a computerized method of creating resumes to examine many values of many variables rather than the often-binary choice sets of resumes prior to her study (see also Lahey and Beasley 2009). Using this revision of the correspondence audit, she could test if employers were less likely to call back older workers due to judgments and assumptions about human capital (statistical discrimination) or due to a general preference for younger workers (taste-based discrimination). She found some evidence for statistical but not taste-based age discrimination. Importantly, her computerized method of creating resumes has also been used to develop several large-scale correspondence audits (e.g., Deming et al. 2016; Oreopoulos 2011). In another study, Leo Kaas and Christian Manger (2012) conducted a correspondence audit in Germany in which they found that Turkish applicants were less likely to receive a callback than German applicants. However, they submitted some applications with two reference letters that included information on personality and work ethic. The authors found that among applications that included these reference letters, there were no statistical differences between the callback rates for German and Turkish applicants, suggesting that employers in Germany engage in statistical discrimination against Turkish applicants. These four studies highlight an important shift in audit studies from simply documenting discrimination to exploring the process in more detail. This trend would continue throughout the following decade and shape the focus and contributions of future audit studies. 10 Rooth makes a distinction that he is specifically testing the combined category. The Current Wave: The Early 2010s to Present Since the early 2010s, the number of audit studies appearing in journals and working paper form has grown exponentially. By my count, the number of audit studies conducted between 2010 and 2017 is already quadruple the number conducted between 2000 and 2009. For that reason alone, it would be incredibly difficult to cover all of these studies with any detail in this section. With apologies to those not covered here, I focus on what I consider to be the most significant developments during the past seven years. However, it is also important to note that researchers have continued to expand the domains of study to areas such as healthcare (Kugelmass 2016; Sharma, Mitra, and Stano 2015; Shin et al. 2016), politics and public service (Butler and Broockman 2011; Einstein and Glick 2017; Giulietti, Tonin, and Vlassopoulos 2015; Hughes et al. 2017; McClendon 2016; White, Nathan, and Faller 2015), religious organizations (Wallace et al. 2012; Wright et al. 2015), eBay and Craigslist transactions (Besbris et al. 2015; Doleac and Stein 2013; Nunley, Owens, and Howard 2011), and new sharing economy market transactions such as Airbnb and Uber (Cui, Li, and Zhang 2017; Edelman, Luca, and Svirsky 2017; Ge et al. 2016). Additionally, researchers have expanded the countries of study to include Argentina (Bóo, Rossi, and Urzúa 2013), Belgium (Baert 2016; Baert and Verhofstadt 2015), Brazil (de Leon and Kim 2016), China (Maurer-Fazio 2012; Maurer-Fazio and Lei 2015; Zhou, Zhang, and Song 2013), the Czech Republic (Bartoš et al. 2016), Ghana (Michelitch 2015), Israel (Ariel et al. 2015; Ruffle and Shtudiner 2015; Zussman 2013), Malaysia (Lee and Khalid 2016), Mexico (Arceo-Gomez and Campos-Vazques 2014; Campos-Vazques and Arceo-Gomez 2015), Norway (Andersson, Jakobsson, and Kotsadam 2012), Peru (Galarza and Yamada 2014, 2017), and Poland (Wysienska-Di Carlo and Karpinski 2014). HUD has also continued to conduct audit studies with a new iteration of the HDS in 2012 (Turner et al. 2013). I believe there have been at least four major developments in audit research during the most recent period: (1) continued attempts to adjudicate among types of discrimination, (2) an increased focus on context and the conditions under which discrimination occurs, (3) an increased focus on methodological issues in audit design, and (4) the inclusion of additional data from outside the audit itself. These developments are not mutually exclusive; many studies incorporate two or more of these developments. Adjudicating among Types of Discrimination Scholars have long sought to understand the reasons for discrimination and to better adjudicate among types of discrimination (Aigner and Cain 1977; Altonji and Blank 1999; Arrow 1972; Becker 1957; Dymski 2006; Gruyan and Charles 2013). Discrimination research has often focused on whether decision makers discriminate based on a general dislike of a certain group (taste-based discrimination) or based on assumptions about the average characteristics of an individual from that group (statistical discrimination). 11 Recent audits have attempted to adjudicate between taste-based and statistical discrimination by varying multiple characteristics and examining differences in response rates between types of characteristics (more or less susceptible to taste-based discrimination) and examining interactions with characteristics that might provide information to overcome statistical discrimination (Agerström et al. 2012; Ahmed, Andersson, and Hammarstedt 2010; Auspurg, Hinz, and Schmid 2017; Baldini and Federici 2011; Bosch, Carnero, and Farré 2010; Capéau et al. 2012; Carlsson and Eriksson 2014; Drydakis 2014; Edo, Jacquemet, and Yannelis 2013; Ewens, Tomlin, Wang 2014; Gneezy, List, 11 David Neumark (forthcoming) provides an excellent review of these and other types of discrimination, so I do not go into more detailed explanation here. and Price 2012; Hanson and Hawley 2014; Hanson and Santas 2014). The results from these studies are somewhat mixed as to whether taste-based or statistical discrimination occurs more often (or some combination of the two). These mixed findings likely stem from the variety of locations and characteristics studied. Two studies related to taste-based versus statistical discrimination stand out among the rest (Bartoš et al. 2016; Pager 2016). In the first, the authors examined how both an individual characteristic, in this case race, and the type of market can lead to “attention discrimination,” or the differential use of available information. The authors set up audits in rental housing and labor markets and found that in the first market, decision makers selected more applicants overall and more often examined additional information from minority applicants. In the later market, decision makers selected fewer applicants overall and more often examined additional information from majority applicants. Thus, discrimination in acquiring information about candidates occurred at the initial stage of selection and varied by the selectivity of the market. We should be cautious to consider how these types of processes – overall response or selection rates in a given market and the differential use of available information – might influence future audits. In the second, Devah Pager (2016) examined whether firms that discriminated in a previous audit are still in business six years later. Economists suggest that an efficient market should eventually weed out taste-based discrimination since not all employers exhibit that type of discrimination and those who do will pay a penalty for inefficient hiring (Arrow 1973; Becker 1957). Using additional data on firm failure, Pager found that prior discrimination is associated with a firm going out of business. Although other factors may explain this relationship, the findings are at least consistent with taste-based discrimination. Context and Conditions Under Which Discrimination Occurs Another major development during this period has been researchers’ increased focus on context and the conditions under which discrimination occurs. Two aspects of context – geographic location and occupation or market characteristics – have played a significant role in recent audits. Those audits that have taken geographic variation into account often examine differences by neighborhood characteristics such as racial, ethnic, immigrant, and SES composition (Acolin, Bostic, Painter 2016; Carlsson and Ericksson 2014, 2015; Carlsson, Reshid, and Rooth 2017; Galster, MacDonald, and Nelson forthcoming; Ghoshal and Gaddis 2015; Hanson and Hawley 2011; MacDonald, Galster, and Dufty-Jones forthcoming). Others have examined geography in more detail by tying discrimination- or prejudice-based theories into the analysis (Besbris et al. 2015, this volume; Gaddis and Ghoshal 2015; Hanson and Hawley 2014; Phillips 2016a). A second strand of research has considered if levels of discrimination are influenced by the types or composition of occupations (Albert, Escot, and Fernandez-Cornejo 2011; Andriessen et al. 2012; Booth and Leigh 2010; Bursell 2014; Carlsson 2011; Derous, Ryan, and Nguyen 2012; Zhou, Zhang, and Song 2013), whether a job is a promotion (Baert, De Pauw, and Deschacht 2016), whether an applicant is overqualified (Baert and Verhaest 2014; Verhaest et al. forthcoming), or market tightness or slackness (Baert et al. 2015; Carlsson, Fumarco, and Rooth 2015; Farber, Silverman, and von Wachter 2017; Vuolo, Uggen, and Lageson 2017a). Some researchers have varied multiple individual characteristics simultaneously and examined interactions to try to capture a broader spectrum of the decision-making process. In particular, recent audits have focused on interactions between race/ethnicity and educational credentials (Carbonaro and Schwarz this volume; Darolia et al. 2015; Deming et al. 2016; Gaddis 2015, 2017d; Lee and Khalid 2016; Nunley et al. 2015), race/ethnicity and criminal record (Ahmed and Lang 2017; Decker et al. 2015; Uggen et al. 2014), race/ethnicity and sexual orientation (Mazziotta, Zerr, and Rohmann 2015) and various combinations of personal characteristics and human capital characteristics (Andersson, Jakobsson, and Kotsadam 2012; Baert and Vujic 2016; Baert et al. 2016b, 2017; Johnson and Lahey 2011; Namingit, Blankenau, and Schwab 2017; Neumark, Burn, and Button 2015; Nunley et al. 2016, 2017; Oreopoulos and Dechief 2012; Pedulla 2016; Phillips 2017). Some of the most interesting research to examine context and conditions has focused on the effects of policies. In one such study, a team of researchers examined whether discrimination against individuals with a disability varied by whether a company was subject to the Americans with Disabilities Act (ADA) (Ameri et al. forthcoming). The authors found that the ADA reduced discrimination against disabled applicants among employers that were covered under the law. A second study used audit and non-audit data to examine differences in age discrimination across states by differences in anti-discrimination policies (Neumark et al. 2017). The authors found no strong relationship between the strength of state laws and discrimination rates. Finally, a third study used a difference-in-differences design with an audit, multiple time points, and a policy change (Agan and Starr 2016). The authors tested the effect of ban-the-box policies, which prevent an employer from collecting information on criminal record, on levels of racial discrimination in hiring. They found that after ban-the-box policies went into effect, levels of racial discrimination increased. The authors suggest that when employers cannot ask about criminal history, they may engage in statistical discrimination and assume that black applicants have a criminal record. Methodological Issues in Audit Design In recent years, scholars have considered at least three methodological issues in audit design: (1) paired vs nonpaired audits, (2) indirect signals of race, and (3) the Heckman critique of unobserved differences between groups. First, in my experience, the question of paired versus non-paired audit design is often a concern during IRB submission and subsequent discussions. A paired audit design opens the research up to an increased chance of experiment discovery because decision makers can potentially see two applicants or inquiries that are very similar. However, conventional wisdom suggests that the paired design is more statistically efficient, decreases the amount of time required for data collection, and can lead to a larger sample size (Lahey and Beasley this volume). In at least two cases, fear of experiment discovery led to a preemptive non-paired audit design (Weichselbaumer 2015, 2016). Additionally, researchers have raised concerns that paired designs may influence findings of discrimination because researchers insert fake applicants into the applicant pool without knowing the composition of that applicant pool (Phillips 2016b; Weichselbaumer 2015). Employers compare applicants to each other and by inserting more than one applicant into a particular pool, researchers may influence the process. In fact, Phillips (2016b) developed a method to test these effects and found that “adjusting for applicant pool composition increases measured discrimination by 20% on average” (2016b: 1). Moreover, proper power analysis suggests that paired audits are not needed as often as researchers think (Vuolo, Uggen, and Lageson 2016, this volume). I have devoted considerable time and effort to a second methodological concern – the indirect signaling of race through names (Gaddis 2017a, 2017b, 2017c, forthcoming). With correspondence audits, researchers lose the ability to directly convey race through appearance and must rely on an indirect signal, such as a name, to signal race. Although prior research occasionally raised some concerns about the signal of names (e.g. Bertrand and Mullainathan 2004), only 17.5% of the studies I reviewed used pretests to examine the perception of names used in an audit (Gaddis forthcoming). My work has shown that racial perceptions of white and black names are often linked with social class (Gaddis forthcoming), Hispanic names are strongly identified (Gaddis 2017a), immigrant generational status can be discerned through names (Gaddis 2017b), and, perhaps most importantly, audit findings are strongly linked to the names researchers use (Gaddis 2017c). Still, more needs to be done to examine the signals we use in audit studies (see next section). The final area of methodological inquiry concerns the Heckman critique of unobserved differences between groups and has received the most scholarly attention of the three issues discussed here (Heckman 1998; Heckman and Siegelman 1993). James Heckman’s critique is that scholars using the audit design assume that unobservable characteristics have equal means across groups, yet scholars cannot confirm that. Heckman suggests that multiple components could enter into the decision-making process – some controlled for by audit design and others unknown to designers but known to the decision makers. In other words, characteristics that researchers do not include on a resume or in an email. These components combine to place a candidate above or below the threshold to receive a response. If the two groups being studied have different variances on these important unobserved components, audit studies may over or underestimate discrimination or detect an effect when there is not one. David Neumark (forthcoming) provides a more detailed discussion of this critique and has devised a method to produce an unbiased estimate of discrimination and avoid this critique (2012). Neumark (2012) reanalyzed Bertrand and Mullainathan’s (2004) original audit data using this method to account for the variance of unobservables and found stronger evidence of racial discrimination. Two individual studies have implemented Neumark’s method, with no clear pattern regarding bias (Baert 2015; Neumark, Burn, and Button 2016). Two other studies have re-analyzed data from multiple audits and suggest that employment audits appear to be susceptible to the Heckman critique (Carlsson, Fumarco, and Rooth 2014; Neumark and Rich 2016). The authors of these two studies advise that scholars still have a lot of work to do in improving the audit method by more directly addressing this critique. Including Additional Data from Outside the Audit A final major development in recent audit research is the inclusion of additional data from outside the audit itself, something done by many of the studies already mentioned in this section. Several researchers have included geographic data on neighborhood and city characteristics to supplement audits (e.g. Acolin, Bostic, Painter 2016; Carlsson and Ericksson 2014, 2015; Ghoshal and Gaddis 2015; Hanson and Hawley 2011). Others have included other types of available data, such as firm closure (Pager 2016), mortgage lender transactions (Hanson, Hawley, and Martin 2017), and existing survey data on racial/ethnic attitudes and beliefs (Carlsson and Eriksson 2017; Carlsson and Rooth 2012). One of the most promising avenues of inquiry into discrimination is the combination of audits with other methods of data collection. Following in the footsteps of Pager and Quillian (2005), researchers are increasingly obtaining a second round of information from the same individuals who previously participated in an audit. Some researchers have followed-up with employers to administer implicit association tests (IATs) to examine the connection between implicit bias and discrimination (Agerström and Rooth 2011; Rooth 2010). Other researchers have followed-up with surveys or interviews after an audit to attempt to better understand the reasons behind discriminatory actions (Bonnet et al. 2016; Midtbøen 2014, 2015, 2016; Zussman 2013). Although institutional review boards (IRBs) may be hesitant to allow researchers to engage in multiple points of contact with audit participants, some researchers have successfully shown that additional methods of data collection do not necessarily need to follow up with the original audit participants (Gaddis and Ghoshal 2017; Kang et al. 2015). I believe that researchers should continue in the direction of the trends discussed above – adjudicating among types of discrimination, focusing on context and the conditions under which discrimination occurs, focusing on methodological issues in audit design, and including additional data from outside audits. In particular, researchers should try to include geographic data in audits, given the wide availability of geographic data and the relative simplicity and usefulness of including such data in analyzing audit outcomes. Next, in the final section, I outline some limitations of correspondence audits and return to the issues discussed in this section with additional thoughts on continuing to improve correspondence audits. LIMITATIONS OF AND WAYS TO IMPROVE CORRESPONDENCE AUDITS Despite the rapid advancement of correspondence audits over the past two decades, several serious limitations exist that scholars must continue to address. Limitations of in-person audits have been covered by others in detail, particularly James Heckman (1998; Heckman and Siegelman 1993), and I draw upon that work here. However, correspondence audits often have their own unique quirks and limitations. By no means is this section intended to be an exhaustive list of all the limitations of correspondence audits, but instead some areas where I see the biggest problems and/or new potential solutions. I highly recommend the reader turn to David Pedulla’s chapter (this volume) for a more extensive and detailed discussion of these and other issues. Perhaps most important is the general limitation of audit studies in uncovering mechanisms rather than simply documenting the existence of discrimination. As discussed in the previous section, recent work has started to expand our knowledge in this area in increasingly innovative ways. Not all questions will lend themselves to design tricks built into studies to help discover mechanisms, nor can researchers always implement complex factorial designs to test potential mechanisms. My recommendation is that researchers should be more open to collecting survey experiment data side-by-side with field data from audit studies (e.g. Diehl et al. 2013; Gaddis and Ghoshal 2017). The deception of the audit study may allow us to document discrimination but a similar scenario presented as a survey experiment may allow us to explore potential mechanisms with the right questions. Moreover, the rise of Amazon’s Mechanical Turk (MTurk) makes collecting survey experiment data relatively quick and cheap (Campbell and Gaddis forthcoming; Porter, Verdery, and Gaddis 2017). In ongoing work combing an audit with a survey experiment, I find that roommate discrimination against many different racial and ethnic groups is driven by issues of cultural fit. However, blacks face higher levels of discrimination than others due to negative perceptions about financial stability and courteousness, despite respondents receiving the same information about all racial/ethnic groups (Gaddis and Ghoshal 2017). These findings would not have come to light if we had implemented a correspondence audit or survey experiment alone. A second major limitation of correspondence audits is indirect signaling of characteristics. Correspondence audits often require signals to be sent through names, statements, lists, or other text embedded in communications. In my own research, I have worked to understand how names can be used to signal race, ethnicity, and immigrant status (Gaddis 2017a, 2017b, forthcoming) and have found that signals of race are conflated with social class and that conflation explains differences in response rates across previous correspondence audits (Gaddis 2017c). Still, more work needs to be done to ensure that construct validity is high when we need to indirectly signal characteristics in correspondence audits. At a minimum, researchers should pretest their signals in a scientific manner to help increase construct validity. Additionally, more work is needed to explore the possibility of alternate signals since there is often more than one way to indirectly signal a characteristic. The signaling of characteristics is also related to the way we can conduct correspondence audits and the level of external validity of those audits. A characteristic such as race or gender may convey different things depending on how it is signaled and the context in which it is signaled. Not only are correspondence audits only as good as the signals they use to convey key characteristics, but audit studies also only tell us about a specific avenue of correspondence with a specific signal. For example, real job seekers may use any combination of online job sites, personal and professional networks, alumni resources, headhunters, and employment events. How race is conveyed and the meaning of race likely vary across these different means of searching for a job. Static, written signals – such as name, professional affiliations, or even checking a box for race – may cue stereotypes about race. Dynamic, interpersonal signals – such as a discussion with a reference or interaction with the individual – may permit more flexibility in thoughts about race. Although others have raised concerns about how audits begin with a narrow sampling frame (e.g., jobs or housing posted in newspapers or on websites) and limit generalizability to the entire job or housing search process (Friedman 2015; Gaddis 2015; Heckman and Siegelman 1993; Pitingolo and Ross 2015), I suggest that the narrow sampling frame also limits our knowledge of discrimination processes only to those that can be conveyed through certain static and often indirect signals. Although in-person audits have occasionally examined multiple outcomes at various stages of the processes they study (Bendick, Jackson, and Reinoso 1994; Pager, Western, and Bonikowski 2009; Turner, Fix, and Struyk 1991), correspondence audits have been almost entirely limited to studying outcomes at the initial contact phase. Critics have pointed out that we do not know whether the disparities witnessed at the initial contact phase lead to disparities at later phases (Heckman 1998; Heckman and Siegelman 1993). Others have used nationally representative data to simulate the effect of employer callback disparities on wages (Lanning 2013). Still, as my own research shows, we should use all the information possible to expand the outcomes examined by audit studies. Additional information in both employment (Gaddis 2015) and housing advertisements (Gaddis and Ghoshal 2015, 2017; Ghoshal and Gaddis 2015) should be used to our advantage. Furthermore, we should consider additional ways that audits might be tweaked to examine other outcomes. In employment audits, do human resources staff visit LinkedIn or Facebook pages, contact references, or attempt multiple contacts with applicants at different rates? Some recent articles provide excellent examples of the directions audits might continue to go in the future (Acquisti and Fong 2015; Baert forthcoming; Bartoš et al. 2016; Blommaert, Coenders, and van Tubergen 2014; Butler and Crabtree forthcoming; and see Crabtree this volume for more discussion). Additionally, is it possible to return to the strategies of earlier audits and use a sub-sample with real humans to proceed deeper into processes, such as sending trained assistants into in-person or Skype interviews? I believe that future waves of audit studies will need to be creative and incorporate more variety in outcomes to push this method forward. THIS VOLUME AND ONLINE RESOURCES This volume is organized into three broad sections: (1) The Theory Behind and History of Audit Studies, (2) The Method of Audit Studies: Design, Implementation, and Analysis, and (3) Nuance in Audit Studies: Context, Mechanisms, and the Future. You are reading the first chapter of the first section and, hopefully, you already have a better understanding of audit studies. In the second chapter, Fran Cherry and Marc Bendick discuss the historical connections between activism and scholarship through audits. Their chapter highlights the potential power of audit studies to not just document discrimination but reduce it as well. The authors advocate for a return to scholar-activism and outline four characteristics that will help facilitate that path. In the third chapter, Stijn Baert provides an excellent overview of labor market correspondence audits conducted since Bertrand and Mullainathan’s groundbreaking study. Baert organizes these studies across two major dimensions: discrimination treatment characteristic, which includes nine federally-banned (U.S.) and five state-banned discrimination grounds, and country of analysis. Overall, the author provides information on 90 labor market correspondence audits across 24 countries. The chapters in the second section give the reader a “behind-the-scenes” look at the nuts and bolts of audit studies, as well as serve as a guide for designing and implementing your own audit studies. In the fourth chapter, Joanna Lahey and Ryan Beasley outline a number of technical aspects related to designing and conducting a correspondence audit. They cover issues of validity, participant selection, timing, technical design of correspondence, matching, sample size, and analysis, among other issues. Their chapter serves as a terrific starting point for anyone needing more information on creating their own audit. In the fifth chapter, Charles Crabtree extends this discussion by providing a detailed overview of designing and implementing an email correspondence audit. He provides information on sample selection, collecting email addresses, sending emails, and collecting outcomes. This chapter is particularly useful in thinking about automating an audit design using programming scripts. A coding appendix for this chapter will be available at auditstudies.com. In the sixth chapter, Mike Vuolo, Christopher Uggen, and Sarah Lageson offer an extensive consideration of matched versus non-matched audit designs. They provide statistical guidelines for when matching is appropriate and show that non-matched audit designs can be more efficient. Additionally, they raise some important substantive points for researchers to think about when deciding to use a matched or non-matched design. Finally, the chapters in the third section provide even deeper insight into the audit process by discussing more design considerations and nuance. In the seventh chapter, William Carbonaro and Jonathan Schwarz outline their thought process in selecting cities in which to conduct an audit, the difficulties of using a small city, the unknowns of the employer side of an audit, and the choice of jobs for a sample. This chapter shares important “lessons learned” from experienced researchers. Although scholars cannot think through all of the possible variables involved in designing and fielding an audit in advance, I think this chapter serves as a great example of how auditing is an incredibly difficult and nuanced process. In the eighth chapter, Max Besbris, Jacob William Faber, Peter Rich, and Patrick Sharkey show how an audit can be designed to investigate a non-individual-level treatment. They use an audit to examine the mechanism of place-based stigma in the relationship between neighborhoods and outcomes for residents of those neighborhoods. Their audit, the discussion of thinking about signaling characteristics, and the theory-based use of geography provide a strong example of what future audits might looks like. In the ninth and final chapter, David Pedulla explores how audits might change and develop in the coming years. He highlights research that identifies mechanisms, examines when and where discrimination happens, and scrutinizes issues of representativeness. David’s chapter serves as a terrific bookend to this volume and should be read closely by anyone wishing to implement an audit of their own. On behalf of the other contributors, we hope you find this volume informative and useful. We have a number of overarching goals for this book: (1) to create a go-to guide for anyone looking to conduct an audit study, (2) to provide resources for using the audit method, both within this book and online, and (3) to record the history of audits. For more information on audits, please consult our website at www.auditstudies.com and take a look at the recommend reading list below. RECOMMENDED READING Comprehensive Articles and Books on Audits “Situation Testing for Employment Discrimination in the United States.” 2007. By Marc Bendick Jr. Horizons Stratégiques, 3:17-39. Clear and Convincing Evidence: Measurement of Discrimination in America. 1993. Edited by Michael Fix and and Raymond J. Struyk. Washington, DC: The Urban Institute. “Experimental Research on Labor Market Discrimination.” Forthcoming. By David Neumark. Journal of Economic Literature. “The Use of Field Experiments for Studies of Employment Discrimination: Contributions, Critiques, and Directions for the Future.” 2007. By Devah Pager. The ANNALS of the American Academy of Political and Social Science, 609:104-33. Reviews of Audits and Discrimination Research “What Have We Learned from Paired Testing in Housing Markets?” 2015. By Sun Jung Oh and John Yinger. Cityscape: A Journal of Policy Development and Research, 17(3):15-59. “The Sociology of Discrimination: Racial Discrimination in Employment, Housing, Credit, and Consumer Markets.” 2008. By Devah Pager and Hana Shepherd. Annual Review of Sociology, 34:181-209. “Field Experiments of Discrimination in the Market Place.” 2002. By Peter A. Riach and Judith Rich. The Economic Journal, 112:F480-F518. “What Do Field Experiments of Discrimination in Markets Tells Us? A Meta-Analysis of Studies Conducted Since 2000.” 2014. By Judith Rich. Available at SSRN: https://ssrn.com/abstract=2517887 “A Multidisciplinary Survey on Discrimination Analysis.” 2013. By Andrea Romei and Salvatore Ruggieri. The Knowledge Engineering Review, 29(5):582-638. Meta-Analyses of Audits “The Persistence of Racial Discrimination: A Meta-Analysis of Field Experiments in Hiring over Time.” Forthcoming. By Lincoln Quillian, Devah Pager, Ole Hexel, and Arnfinn Midtbøen. Proceedings of the National Academy of Sciences. “Ethnic Discrimination in Hiring Decisions: A Meta-Analysis of Correspondence Tests 1990-2015.” 2016. By Eva Zschirnt and Didier Ruedin. Journal of Ethnic and Migration Studies, 42(7):1115-34. Articles and Books on the Methodology of Audits, Discrimination, and Field Experiments Field Experiments (General) “Field Experiments Across the Social Sciences.” 2017. By Delia Baldassarri and Maria Abascal. Annual Review of Sociology, 43:41-73. Field Experiments: Design, Analysis ,and Interpretation. 2012. By Alan S. Gerber and Donald P. Green. New York, NY: W.W. Norton. “The Principles of Experimental Design and Their Application in Sociology.” 2013. By Michelle Jackson and D. R. Cox. Annual Review of Sociology, 39:27-49. Audits (General) Audit Studies: Behind the Scenes with Theory, Method, and Nuance. 2017. Edited by S. Michael Gaddis. Springer. Discrimination (General) Measuring Racial Discrimination. 2004. By Rebecca N. Blank, Marilyn Dabady, and Constance F. Citro. Washington, DC: The National Academies Press. Automating Resume Creation for Audits “Computerizing Audit Studies.” 2009. By Joanna N. Lahey and Ryan A. Beasley. Journal of Economic Behavior & Organization, 70(3):508-14. Critiques of Audits and Solutions “Detecting Discrimination.” 1998. By James J. Heckman. Journal of Economic Perspectives, 12(2):101-16. “The Urban Institute Audit Studies: Their Methods and Findings.” 1993. By James J. Heckman and Peter Siegelman. In Clear and Convincing Evidence: Measurement of Discrimination in America, edited by M. Fix and R. J. Struyk, 187-258. Washington, DC: The Urban Institute Press. “Detecting Discrimination in Audit and Correspondence Studies.” 2012. By David Neumark. The Journal of Human Resources, 47(4):1128-57. “Do Field Experiments on Labor and Housing Markets Overstate Discrimination? A Re-Examination of the Evidence.”2016. By David Neumark and Judith Rich. Available at NBER: http://www.nber.org/papers/w22278 Signaling Characteristics in Audits “How Black are Lakisha and Jamal? Racial Perceptions from Names Used in Correspondence Audit Studies.” Forthcoming. By S. Michael Gaddis. Sociological Science. “Racial/Ethnic Perceptions from Hispanic Names: Selecting Names to Test for Discrimination.” 2017. By S. Michael Gaddis. Available at SSRN: https://ssrn.com/abstract=2975829 “Assessing Immigrant Generational Status from Names: Scientific Evidence for Experiments.” 2017. By S. Michael Gaddis. Available at SSRN: https://ssrn.com/abstract=3022217 “Auditing Audit Studies: The Effects of Name Perception and Selection on Social Science Measurement of Racial Discrimination.” 2017. By S. Michael Gaddis. Available at SSRN: https://ssrn.com/abstract=3022207 Statistical Analysis of Audits “Statistical Power in Experimental Audit Studies: Cautions and Calculations for Matched Tests with Nominal Outcomes.” 2016. By Mike Vuolo, Christopher Uggen, and Sarah Lageson. Sociological Methods & Research, 45(2):260-303. Theoretical Articles and Books on Discrimination “Taste-Based or Statistical Discrimination: The Economics of Discrimination Returns to its Roots.” 2013. By Jonathan Guryan and Kerwin Kofi Charles. The Economic Journal, 123:F417-32. Theorizing Discrimination in an Era of Contested Prejudice: Discrimination in the United States, Volume 1. 2008. By Samuel Roundfield Lucas. Philadelphia, PA: Temple University Press. REFERENCES Acolin, Arthur, Raphael Bostic, and Gary Painter. 2016. “A Field Study of Rental Market Discrimination across Origins in France.” Journal of Urban Economics, 95:49-63. Acquisti, Alessandro, and Christina M. 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PLOS ONE RESEARCH ARTICLE Central bank communication, shadow banking, and bank risk-taking: Theoretical model and PVAR empirical evidence Jing Zhang☯, Shuai Chen☯, Honglei Liu ID* School of Finance, Shandong University of Finance and Economics, Jinan, Shandong, P.R. China a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Zhang J, Chen S, Liu H (2022) Central bank communication, shadow banking, and bank risk-taking: Theoretical model and PVAR empirical evidence. PLoS ONE 17(9): e0275110. https://doi. org/10.1371/journal.pone.0275110 Editor: Haijun Yang, BeiHang University School of Economics and Management, CHINA Received: May 20, 2022 Accepted: September 10, 2022 Published: September 28, 2022 Copyright: © 2022 Zhang 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. ☯ These authors contributed equally to this work. * reneeliuhonglei@163.com Abstract The rapid development of Chinese shadow banking, which relies on the banking system, significantly impacts nonneutral bank risk-taking. Central bank communication also plays a vital role in risk prevention and mitigation in the banking system in a modern central banking system. To address the issues of central bank communication, shadow banking, and bank risk-taking, we construct a new theoretical model that includes shadow banking based on the M-S model. Based on the central bank written communication index measured by the Monetary Policy Implementation Report 2009–2019 and balanced panel data of 35 listed banks, we design an empirical PVAR model. This study finds that central bank communication regarding domestic easing economy and policy positively affects bank risk-taking, while central bank communication and mechanism exists in China. Moreover, the growth rate of shadow banking size also positively encourages bank risk-taking. Furthermore, central bank communication regarding foreign policy is negatively related to bank risk-taking in the robustness test, supporting the above findings. The analysis of equity heterogeneity shows that the positive effect of central bank communication on bank risk-taking in shadow banking is more pronounced in small and medium-sized shareholdings. Further analyzing the economic consequences of central bank communication and shadow banking on bank risk-taking, we find that banks’ risk-taking positively affects their share of noninterest income and total asset size. Data Availability Statement: All files are available from the official website of the People’s Bank of China (http://www.pbc.gov.cn/) and WIND database (https://www.wind.com.cn/). 1. Introduction Funding: This paper was supported by a grant of the National Natural Science Foundation of China under the project entitled "Shadow Banking, Credit Transmission and Asymmetric Effects of Monetary Policy" (Grant No. 71573156); Shandong federation of social science (2022-JRZZ-06); the Natural Science Foundation of Hebei Province (G2020208002); and Humanities and Social Nonneutral bank risk-taking has been of great importance to countries since it surfaced. The experience of the global crisis shows that the impact of financial innovation, typically represented by shadow banking, on banks’ risk-taking cannot be underestimated. The same period proposed a monetary policy bank risk-taking channel and focused on the continued easing of monetary policy and bank risk-taking. However, national monetary policy in recent years has shown a new trend of increasingly frequent central bank communication operations and increasingly prominent roles. PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 1 / 29 PLOS ONE Science Research Project of Hebei Education Department (SQ2021138, SQ201106). Competing interests: The authors have declared that no competing interests exist. Central bank communication, shadow banking, and bank risk-taking Establishing a modern central banking system worldwide is inseparable from a perfect central bank communication system. Automating and rationalizing monetary policy communication guides market participants’ expectations and facilitates improving the transmission mechanism of monetary policy. The People’s Bank of China has been taking the initiative to strengthen communication since 2020, gradually forming institutionalized communication methods such as announcing operational arrangements for medium-term lending facilities in advance and issuing press releases on policies. However, the long-established, institutionalized, authoritative, widely influential, and operationally independent communication method remains the quarterly mid-monthly release of the Report on the Implementation of China’s Monetary Policy (hereafter referred to as the Report). The Report received much attention. Many studies have clarified its essential impact on financial markets, business investment, and inflation expectations, affirming its positive role in the transmission mechanism of monetary policy [1, 2]. Central bank communication also play an essential role in preventing and defusing major systemic risks by influencing banks’ risk-taking. Unfortunately, this has not received enough attention from the academic community, as evidenced by the lack of research on central bank communication and bank risk-taking in China. However, the People’s Bank of China has continued to combat some of the irregular operational behaviors on the deposit side of banks since the fourth quarter of 2020 at the policy practice level. They use various information and communication methods to guide the entity’s interest rate expectations and influence asset-side decisions. Central bank communication naturally has a direct impact on the risktaking appetite of banks. Therefore, the development of the current situation needs to be closely followed by academic research. It is necessary to carry out theoretical and empirical investigations on the central bank’s communication behavior concerning banks’ risk-taking. However, shadow banking is closely related to risk-taking in banks, especially in China, where the relationship is even closer. Scholarly studies note that the new regulatory restrictions introduced by the regulatory authorities in China have facilitated the further development of shadow banking in banks in the context of the rapid growth of fintech [3]. Shadow businesses helps banks do away with their restrictions. They obtain more sources of funds through offbalance-sheet wealth management and deposit-like products and diversify the use of funds through asset securitization and channel-type businesses. These shadow banking behaviors are profoundly linked to banks’ risk-taking. The off-balance sheet operations in the U.S. banking industry have continued to increase the proportion of nondeposit liabilities and noninterest income [4]. This paper focuses on the theoretical and empirical investigation of the threefold relationship between central bank communication, shadow banking, and bank risk-taking, with the following main contributions. First, this study builds on the literature on the impact of central bank communication on bank risk-taking using the M-S theoretical model by incorporating shadow banking factors, and deriving an analysis of the important role of both central bank communication and shadow banking on bank risk-taking. We slightly expand the research on the effects of central bank communication as a policy tool, as well as the mechanism of central bank communication and exchange in the bank risk-taking channel of monetary policy, trying to touch the currently undiscovered marginal gap in theoretical research between central bank communication, shadow banking, and bank risk-taking. Second, the impact of central bank communication and shadow banking on bank risk-taking is not transient. The dynamic relationship between the three may be of more interest, so this paper uses the PVAR empirical method to test the long-term dynamic relationship. Third, banks with different ownership systems may not be as sensitive to central bank communication and may not be affected by shadow banking to the same extent, so this paper considers the effect of ownership heterogeneity. Fourth, what long-term economic consequences might be expected of central bank PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 2 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking communication and shadow banking-influenced bank risk-taking? This paper explores the impact of bank risk-taking in the above endogenous system on bank profitability and asset size. Finally, this paper constructs a written communication index of the Chinese central bank on the domestic economy and policies and a written communication index of the Chinese central bank on foreign economies and policies, which to some extent provides a basis for the Chinese policy authorities to make decisions. The rest of the paper is organized as follows. Section 2 presents the literature review. Section 3 constructs a novel economic model. Section 4 provides a brief description of our data sources. Section 5 reports our main empirical results, and Section 6 provides further analysis. Section 7 concludes. 2. Literature review 2.1. Unconventional monetary policy and bank risk-taking Existing studies have confirmed the existence of a precrisis monetary policy bank risk-taking channel at the theoretical level [5]. At the empirical level [6, 7], this means that low-interest rates from accommodative monetary policy cause a rise in banks’ risk-taking. This type of research focuses on the impact of traditional monetary policy instruments on banks’ risk-taking. However, low-interest rates in many developed economies have been maintained for almost a decade after the financial crisis. They are expected to continue, with particular attention to the fact that central bank monetary policy has different effects during periods of financial stress compared with normal periods [8]. Some scholars have focused on the importance of unconventional monetary policy instruments by central banks in the zero interest rate floor dilemma and bank risk-taking. Brana, Campmas, and Lapteacru [8] use a dynamic panel model of threshold effects to study the impact of monetary policy, particularly unconventional monetary policy, on bank risk-taking behavior in Europe over the period 2000 to 2015. They find that low- interest rates and increased central bank liquidity through accommodative monetary policy have a nonlinear deleterious effect on bank risk. Matthys, Meuleman and Vennetb [9] used bank-firm level corporate syndicated loan data to construct a VAR model to identify bank risk-taking due to unconventional monetary policies in the U.S. from 2008 to 2015. An accommodative monetary environment was associated with overall lower loan spreads. 2.2. Central bank communication and bank risk-taking The predominance of central bank communication in the unconventional monetary policies of national central banks has attracted the attention of scholars. They have launched a deep inquiry into the effectiveness of central bank communication and a communication approach based on clarity and transparency [10–12], tone of voice mood [13–15], language style [16], and verbal and written tools [17, 18]. Among them, written forms of communication such as the Monetary Policy Implementation Report, the Financial Stability Report, and the Financial Statistics Report, and press releases have received much attention and research, mainly around how to guide interest rate expectations [17, 19–21], exchange rate expectations [22], and inflation expectations [23]. In particular, a large number of studies have addressed the effects of financial market stability [2], financial asset prices [1, 19], and financial asset returns [20, 21, 24, 25]. In addition, a small number of scholars have started to study the impact of firms [26] or macroeconomic operations [18, 27]. Some scholars have also considered central bank communication’s monetary policy transmission perspective. However, existing studies mainly focus on traditional transmission channels [22, 28, 29] and cross-country spillover transmission channels. There is less empirical evidence involving bank risk-taking channels. However, since Borio and Zhu [5] proposed the bank risk-taking channel, they have identified central PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 3 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking bank communication and the communication mechanism as one of the three main mechanisms of action, affecting banks’ risk-taking from a theoretical and practical point of view. Blinder [30] was the first to show the transparency effect of central bank communication and exchange. In other words, it help banks more accurately predict the behavior of the monetary authority, reducing the risk premium while increasing bank risk-taking. Borio and Zhu [5] also proposed the "insurance effect" of central bank communication and exchange based on the above. The central bank’s reaction function can transmit the asymmetric impact of interest rate changes, which can effectively block the downside risk of the economy and reduce bank risk-taking. Later, based on the theory constructed by previous authors, Li and Gao [31] introduced the M-S model of Morris & Shin [32] (a theoretical model of public information affecting social welfare) to derive a proof of the effect of central bank disclosure on commercial banks’ expectations. They pointed out that central bank communication positively affects banks’ risk preferences. 2.3. Shadow banking and bank risk-taking In summary, it is clear that central bank communication, which naturally exists with the creation of central banks, has received great attention in the last decade or so and is seen as an innovative and unconventional monetary policy tool [30]. A major innovation in the financial sector at the same time as central bank communication is shadow banking. Due to the financial crisis caused by persistently loose monetary policy, shadow banking and bank risktaking channels have received widespread attention. There is a thought-provoking and nuanced relationship between central bank communications, shadow banking, and bank risk-taking [5]. Scholars were the first to consider how shadow banking affects the effectiveness of monetary policy. Banks’ existence of off-balance-sheet shadow banking complicates the transmission of monetary policy. The shadow bank is already in a black box, affecting the traditional transmission mechanisms such as monetary and credit channels of monetary policy [33, 34], impacting the traditional monetary multiplier theory and increasing the difficulty of economic regulation by monetary authorities [35, 36]. Of course, the fact that shadow banking interfered with the risk nonneutrality of banks before the 2008 financial crisis and played an important driving role in the overall risk-taking channel has led to skepticism [37]. After the crisis, the impact of shadow banking on bank risk-taking has been controversial but still valued [38, 39]. Some literature has examined shadow banking as a major factor and how it affects commercial banks’ risk-taking. There is also literature on shadow banking as a moderating factor of the impact of traditional monetary policy instruments on commercial bank risk-taking. Existing studies have focused on the impact of traditional monetary policy tools on bank risk-taking. The study of the effectiveness of nonbank subjects of central bank communication and shadow banking and traditional channels of monetary policy, with significant academic contributions, is the logical starting point and theoretical basis of this paper. At the same time, few current studies include central bank communication, shadow banking, and bank risk-taking in the same logical and analytical framework for a detailed and in-depth discussion. This paper attempts to clarify the relationship between the three by conducting an in-depth study of the impact of central bank communication and shadow banking on bank risk-taking. Based on the M-S model of Morris et al. [32], we construct a novel model that includes central bank communication, shadow banking, and bank risk-taking. We propose hypotheses based on this model, measure the written communication index of central bank communication using the word extraction method, combine the data of 35 listed banks in China, and use the PVAR model to carry out an empirical test. PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 4 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking 3. Economic model Both central bank communication and shadow banking affect the risk-taking of commercial banks. For this reason, we draw on Keynes’s "beauty contest" idea, and based on the M-S model of the impact of public information on social welfare by Morris et al. [32], we introduce shadow banking to construct a new model. We theoretically explore the relationship between a central bank’s written communication, shadow banking, and banks’ risk-taking and propose corresponding research hypotheses based on the derivation results of the theoretical model. First, consider all banks as a whole and assume that banks are uniformly distributed in the interval [0,1], pi is the preference of bank i for risk, p is the preference of banks as a whole for R1 risk, and p� � 0 pj dj is the average risk preference of banks as a whole. Qi is the selection differR1 ence between bank i and bank j, and Qi � 0 ðpi pj Þ 2 dj; Q�i is the average selection differR1 ence of all banks, and Q�i ¼ Qj dj. υ is the basic economic state. 0 The utility of bank i is composed of two parts: the first is the distance between bank i’s risk appetite pi and the underlying economic state υ, which is called "judgmental welfare loss"; and the second is the distance between bank i’s choice Qi and bank’s average choice Q�i , which is called the "coordinated welfare loss". Banks i judge the weight of the two parts according to the externality, assuming that the weight of the coordinated welfare loss is λ, then the weight of the judged welfare loss part is 1 − λ, the larger λ the greater the externality, and the existence of externalities causes social inefficiency. At the same time, we assume that the externality is a zero-sum game, which means the gain for the winner is the cost for the loser. Consequently, the utility function of bank i is expressed as follows. ui ðp; vÞ ¼ ð1 l Þ ðp i vÞ 2 lðQi �Þ Q ð1Þ Clearly, the utility of the entire banking system is defined as the average of the (normalized) utility of each bank, denoted as Z 1 Z 1 1 2 Wðp; vÞ ¼ ui ðp; vÞdi ¼ ðpi vÞ di ð2Þ 1 l 0 0 The central bank is concerned with the utility of the entire banking system, so it will strive to make the actions of all individual banks as close as possible to the state of the entire economy υ. On the other hand, bank i pursues its own utility maximizing first-order conditions, as reflected by pi � ¼ ð1 lÞEi ðuÞ þ lEi ðp�Þ ð3Þ R1 where p� � 0 pj dj , is the average risk appetite of the banking system, and Ei(�) is the expectation of υ and p� by banks after obtaining the information communicated by the central bank. If the underlying economic state υ is known, then for all banks, pi = υ, utility is maximized at economic equilibrium, and there is no conflict between individual rationality and overall banking system optimality. The reality, however, is that the basic state of the economy in the intricate economic and financial environment υ is uncertain. Thus, we consider the case of economic state υ uncertainty. At this point, banks have access to public information since public information is available to all banks, denoted here by y. y¼uþZ ð4Þ When shadow banking is not considered, η is a bias in the central bank’s understanding of PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 5 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking the underlying economic state. η obeys a normal distribution with variance s2Z and the precision of the central bank communication is a ¼ s12 . Z In addition to the central bank’s public information, bank i also obtains private information, which is assumed not to be available to other banks, denoted by xi. xi ¼ u þ xi ð5Þ where ξi is independent of υ, η. When shadow banking effects are not considered, ξi obeys a normal distribution with variance s2xi and private information precision b ¼ s12 . xi We consider the existence of a shadow banking situation. The emergence of shadow banking in China stems from bank deposit-to-loan ratio restrictions. Like foreign shadow banks, they are innovative tools for off-balance-sheeting banks’ high-risk assets, which can lead to ever-increasing real leverage of the banking system. It is evident that while shadow banking helps banks pursue high profits, it also leads to a continuous accumulation of risks [40]. More importantly, multiple nested and structured shadow banking products hide much important information about the banking system and are extremely opaque. They are outside the supervision of the monetary authorities, and central banks have increased difficulties in capturing bank risks and the current state of the economy. It can be seen that the existence of shadow banks increases the difficulty of the central bank in grasping the basic economic state, resulting in a deviation of the central bank’s understanding of the basic economic state from η to τ and the variance increases from s2Z to s2t . Then, the accuracy of central bank communication becomes as ¼ s12 , which is smaller than the communication accuracy a ¼ s12 when shadow t Z banks are not considered. Here, assuming that s represents the shadow bank, as ¼ s12 ¼ sa ¼ ss2 ; 0 < s < 1, and so the public information after considering the shadow t Z bank is: ys ¼ u þ t ð6Þ Due to Chinese banks’ unique historical background and institutional characteristics, they are susceptible to central bank information but reflect little in their own information communication. Meanwhile, Chinese shadow banking is essentially a shadow banking system and a linear extension of banks’ traditional lending business [41]. Therefore, we do not change the assumption of the private information of banks and argue that shadow banking happens to be a reasonable means for banks to respond to the central bank’s communication and regulation. Based on the above analysis, the risk appetite p�i ðxi ; ys Þ is chosen by bank i when faced with public information ys = υ + τ and private information xi = υ + ξi. Therefore, the conditional expectation of bank i for the basic economic state υ and the private information of other bank j is expressed as � � bxi þ as ys Ei ðujxi ; ys Þ ¼ Ei xj jxi ; ys ¼ as þ b ð7Þ Drawing on Cornand and Heinemann [42], the economy as a whole is assumed to face a linear information function. pi ðxj ; ys Þ ¼ kxj þ ð1 PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 kÞ ys ð8Þ 6 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking Then the optimal risk appetite of banks is pi ðxi ; ys Þ ¼ kxi þ ð1 k Þ ys ð9Þ The bank’s estimate of the average optimal risk appetite of other banks is � � as ys þ bxi � E i ðp Þ ¼ k þ ð 1 k Þ ys as þ b ð10Þ Substituting Eqs (7) and (10) into Eq (3), we obtain pi � ðxi ; ys Þ ¼ ð1 � ¼ ð1 lÞ as ys þ bxi as þ b � lÞEi ðvÞ þ lEi ðp�Þ �� � � kb þl xi þ 1 as þ b � � � lk þ 1 l ¼b xi þ 1 as þ b � � kb y as þ b s � lk þ 1 l b ys as þ b ð11Þ By comparing the coefficients of Eqs (9) and (11), we can see that k¼ bðlk þ 1 lÞ as þ b ð12Þ k¼ bð1 lÞ as þ bð1 lÞ ð13Þ Solving for this, we obtain Then the optimal risk appetite chosen by bank i is pi � ðxi ; ys Þ ¼ �ðys Þ ¼ bð1 lÞ as x þ as þ bð1 lÞ i as þ bð1 lÞ ys ð14Þ Derivation of the central bank communication information ys in the presence of shadow banking yields @�ðys Þ as ¼ @ys as þ bð1 lÞ >0 ð15Þ Based on the above theoretical model derivation, the following hypothesis is proposed. Hypothesis 1: Central bank communication positively affects banks’ risk-taking preferences; the more adequate the central bank’s information communication, the greater banks’ risktaking preferences. Derivation of the shadow bank s in Eq (14) yields @�ðys Þ bð1 lÞ ðys xi Þ ¼h i2 @s s þ bð1 lÞ s2Z s2 ð16Þ Z Eq (16) shows that the relationship between bank risk-taking and shadow banking depends on the degree of public and private information saturation. The information conveyed by the central bank is of general interest and market participants extract useful information from it on which to base their decisions. PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 7 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking In the short term, it is often found in theory and reality that central banks do not grasp current economic conditions well [43]. The accuracy of central bank communication is influenced by a number of factors such as market conditions, national contingencies, specific dates or deadlines [44], and deliberate failure to inform. At the same time, there is ambiguity in central bank communications, with the pursuit of too many objectives leading to inconsistent communications and conflicting expressions in interperiod central bank communications. It is evident that imperfect information to assess the basis of central bank decisions or monetary policy can lead to complications in the private sector’s response to central bank policy [45]. There are forecasting and credit behavior frictions in banks’ communication of information to the central bank [46], and it is very difficult to determine the short-term transparency and information content of the central bank [47]. In other words, in the short term central bank communication cannot improve the accuracy of market participants’ forecasts. In contrast, commercial banks generally have a relatively greater grasp and saturation of their own information over a shorter period, especially in the early stages of carrying out shadow banking, where the technical means are relatively simple and commercial banks can control them more easily. Thus ðys xi Þ < 0, and @�ðys Þ bð1 lÞ ðys xi Þ ¼h i2 < 0 @s s þ b ð 1 l Þ s2Z 2 s ð17Þ Z In the long run, forward-looking guidance on central bank communication has sustained predictive power [48]. Monetary policy indicators such as interest rates evolve along the path the central bank wishes [1]. Central bank communication indicators are three months ahead of economic conditions [12], and financial markets are vulnerable to central bank communication of long-term objectives [49]. Central bank communication can greatly influence monetary policy expectations, changing risk premia to shape long-term interest rates [50]. Central bank communication cannot reduce the divergence of expectations of market participants in the short term, but it is indeed very effective in the long term [44]. As a key player in the financial markets and a key hub for information collection and processing, banks will continue to digest and absorb central bank public information and endogenously adjust their future expectations of the path of monetary policy in light of new economic developments as external information becomes more certain. Over a long period, the dynamics of banks’ business decisions and the overall situation will be more complex, especially as it will be difficult to fully and effectively capture the technical complexity and measurement difficulties of shadow banking. Through constant innovation and nesting, shadow banking has created an extraordinarily complex and difficult process for clarifying the structure, resulting in a large amount of idle funds. Coupled with the incentive to avoid regulation, shadow banking exploded [51]. The subprime crisis is a good example of the inability of commercial banks to grasp the information about themselves that underlies shadow banking. The existence of shadow banking makes it difficult to clearly access and measure individual information such as the efficiency and profitability of banks, the accumulation of risks in the financial system and information on deep individual environmental decisions such as the real economy and national financial stability [52]. This seriously reduces the transparency of banks’ balance sheets [53]. Therefore, in the long run, the external public information communicated by the central bank is more certain for the individual bank, while its own internal private information is less PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 8 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking precise, so that ðys xi Þ > 0, when @�ðys Þ bð1 lÞ ðys xi Þ ¼h i2 > 0 @s s þ b ð 1 l Þ s2Z s2 ð18Þ Z Therefore, we propose the following hypothesis. Hypothesis 2: The existence of shadow banking in the context of the release of public information by the central bank is beneficial to banks in the short run to reduce risk-taking appetite but may lead to higher risk-taking by banks in the long run. Another point is that although some studies have argued that the monetary policy transmission mechanism is overall similar across countries [54], there is still variability. State-owned and large banks in developed economies are more closely related and more sensitive to monetary policy [55]. However, Chinese scholars have found that large state-owned banks’ (Bank of China, Agricultural Bank, Industrial and Commercial Bank, Construction Bank and Postal Reserve Bank) monetary policy transmission effects are not ideal [36]. Ma and Wang [56] attribute this phenomenon to the difference in financial systems. The dominant financial systems in developed countries such as the UK and the U.S. are the financial markets. In China, however, it is banks, while the regulatory system of the Chinese banking sector (deposit to loan ratio, legal reserve, and capital adequacy ratio) is strict and well established. As a result, the size of loans is subject to many restrictions, which may weaken the transmission effect of monetary policy. In addition, when we consider the issue of banks in China, a point that cannot be circumvented is the size of banks and the nature of their property rights. In China, unlike small and medium-sized joint-stock banks, the more specific nature of state-controlled property rights results in large state-owned banks having the following characteristics. ① The pursuit of profit is less motivated and more focused on safe and sound operations. Unlike small and medium-sized joint-stock banks that constantly pursue profit maximization goals and relatively short-term appraisal mechanisms, large state-owned banks are not intrinsically driven by the pursuit of profit. Sound and safe operations are their bottom line, which determines bank leaders’ promotion, and relegation and career development [36]. Safety bottom-line thinking has led large state-owned banks to be relatively risk-averse and inclined to lower risk-taking. ② Banks implement national policies and are not sensitive enough to the information communicated by the central bank. Large state-owned banks have high asset and liability ratios, with funds flowing to enterprises of a state-owned nature. In particular, large state-owned enterprises’ capital use compliance and rigidity make large state-owned commercial banks insensitive to loose policies and central bank communication. However, small and medium-sized banks have relatively low gearing ratios, and their intrinsic profit motive drive also requires them to expand their business scale. Private enterprises are the main subjects of their fund utilization. Based on relatively single domestic financing channels, small and medium-sized banks have more substantial bargaining power over private enterprises. However, at the same time, they must pay close attention to the policy guidance of the monetary authorities. In other words, they are more sensitive to central bank communication and exchange. ③ The banking system is in an important position, and the central bank’s communication has given more consideration to the stakes. Large state-owned banks are highly susceptible to risk transmission to the entire economic system as they are systemically important banks. PBOC communication has considered the policy’s possible impact on them. Nevertheless, systemic risks are unlikely to arise for city and rural banks. Small and medium-sized joint-stock banks’ communication functions are less considered by small and PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 9 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking medium-sized joint-stock banks, which may have a greater impact on risk-taking than the central bank’s communication. Limited operating areas, relatively fixed sources of funding, and limited ability to place funds, coupled with rigid deposit-to-loan ratio regulation, force small and medium-sized joint-stock banks to be more proactive in shadow banking [36, 57], all of which cause a rise in bank risk-taking and greater sensitivity to central bank communication. Hypothesis 3: The impact of central bank communication on the risk-taking of small and medium-sized joint-stock banks is more pronounced in shadow banking than large stateowned banks. 4. Empirical model and data 4.1. Empirical design In the literature, VAR models have been widely used in studying monetary policy transmission channels [58], and the research involved in the risk-taking channel is no exception [59]. Although VAR models are useful for addressing the linkages between monetary policy and economic agents and do not rely on economic theory as much as structural equation models do, they suffer from the "curse of dimensionality", meaning they cannot be applied to panel data. Therefore, some scholars have expanded their thinking and introduced the panel PVAR model into the research of bank risk-taking [60]. Considering the research topic of this paper and the fact that the PVAR model is very suitable for short time series, this paper uses this model for empirical testing. Based on the study, we developed the following main regression model. Xp Yit ¼ ait þ A Y þ dt þ fi þ εit ð19Þ j¼1 j i;t j i 2 f1; 2; . . . . . . ; Ng; t 2 f1; 2; . . . . . . ; Tg where Yit is a 1 × k dimensional vector of five variables at year t of the i-th bank, expanded as follows: 0 EPE 1 t B C B SB C B t C B C Yit ¼ B CIt C B C B C @ BRTit A ð20Þ RISKit where EPE represents macroeconomic conditions, policy environment (interest rates, economic conditions, price level), S.B. represents shadow banking size variables, CI represents central bank communication index, BRT represents bank-level factors, RISK represents bank risk-taking, ait represents the longitudinal intercept, Aj is the parameter matrix, j represents the lag order of the variable, dt is the time effect vector of the PVAR model, fi is the individual fixed effect of the PVAR model, and εit represents the random disturbance term of the model. This variable order draws on Buch, Eickmeier and Prieto [61] and Zhao and He [60], where macro variables are placed before micro variables. For the order of the two variables of shadow banking and central bank communication, a robustness test of randomly changing the order follows. PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 10 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking 4.2. Variables and data description 4.2.1. Building a central bank communication index. Central bank communication is how the central bank announces information on monetary policy objectives, intermediary indicators, operational indicators, and monetary policy tools to market participants. The literature often uses the wording extraction method in document analysis to extract, filter, and integrate critical statements from some public reports of the central bank as indicators of central bank communication. Because the forms of written communication such as the "Monetary Policy Implementation Report" and the "Resolution of the Monetary Policy Committee Meeting" are relatively fixed and more formal and authoritative, they have become the main objects of the word extraction method. Moreover, the word extraction method is objective and systematic, so this paper also does the same. We mainly adopt the word extraction method for the "China Monetary Policy Implementation Report" (hereinafter referred to as the "Report"). We draw on the methods used by Heinemann and Ullrich [13], Bian and Zhang [62], and Gao and He [29] to construct the central bank’s communication index (cinei, hereinafter referred to as the central bank communication index) for the domestic macroeconomic and policy situation as a proxy variable for central bank communication. This paper considers that China’s commercial banks occupy an important position in the world. According to the 2022 "The Banker" Global Banking Brand Value Ranking, 20 banks in China have entered the world’s top 100 banks, and 48 banks have entered the world’s list of the top 500 banks. In addition, the business scope of Chinese commercial banks is transnationalized and globalized, and their risktaking is closely related to the global economic situation and policy situation. Therefore, this paper innovatively constructs the central bank’s communication index (ciwai), on foreign macroeconomic and policy conditions as a robustness test variable for central bank communication. The People’s Bank of China (PBOC) has been publishing the Report on the PBOC official website quarterly since 2001, which provides an in-depth analysis of the macroeconomic and financial situation, explains monetary policy operations, and discloses the next monetary policy orientation. It is the core channel of communication between the PBOC and market players. In order to avoid the problem of duplication, we only select the text part for wording statistics. In order to ensure the validity of the statistics, we exclude the extreme cases that may be caused by major black swan events, such as the financial crisis in 2008 and coronavirus epidemic in 2020. The People’s Bank of China formally introduced "expectation management" in 2009, so we selected 44 issues from the first quarter of 2009 to the fourth quarter of 2019. The text of the Report is used as the basis to construct the central bank’s communication index using the following. We extracted keywording from the Report. From the 44 issues of the Report, the People’s Bank of China (PBOC) selected the key terms of the domestic and foreign policy environment (easing, moderate easing, steady, tightening, and tightening), economic development (overheating, rising, stable, falling and recession), price trend (inflation risk, rising, stable, falling and deflation), liquidity situation (excess, abundant, moderate, insufficient and shrinking), employment level (high, good, moderate, falling and low) and real estate market situation (overheating, rising, stable, falling and recession). The frequency of each phrase and related descriptions in each issue of the Report (see Table 1) is counted as the raw data basis for constructing the central bank’s domestic (cinei) and foreign (ciwai) communication index. We construct a central bank communication index based on an a priori formula. Based on the information shown in Table 1, the study constructs the central bank disclosure index Formula (14) according to Heinemann and Ullrich [13] and Wang et al. [26], and the statistical PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 11 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking Table 1. Specific expressions of the wording extracted from the monetary policy implementation report. Phrase Type Subdivision Assignment value Policy Environment Easing policy 1 Moderate easing policy 0.5 Macroeconomic conditions Detailed Expression Easing Policy. Active Fiscal Policy, Investment Policy, Expanding Domestic Demand, Growth, Capital Support Policy, Foreign Trade Policy, Tax Reduction, Policy Revenue Reduction, Stimulus (Consumption) Policy, Financial Policy Support Moderate, Reasonably Lenient, Controlled Stable Policy 0 Moderate Austerity -0.5 Robust, Neutral, Moderate Austerity -1 Moderate Tightening Economic overheating 1 Overheated and Fast-Growing Economy Economic Growth 0.5 Tightening And Tightening of Policies Fast Economic Growth Stable (Restorative) Economic Growth, Rebound, Steady Development, Positive Economic Situation, Improving Fundamentals Economic (Heat) Expectations Index Up, Confidence Up Economic Stability 0 Economic decline -0.5 Stable, Healthy, Orderly, Recovery, Stabilization, Easing Cold Economy, Gloomy Outlook Economic Growth Decelerates, Declines, Slumps, Slows Price level Liquidity level Full employment level Recession -1 Deterioration, Recession, Weakness Inflation Risk 1 Excessive Price Increases, Inflation Risk Price increases 0.5 Price stability 0 Decrease in prices -0.5 Inflationary Pressure, Upward (Rising) Price Pressure Stable, Stable Prices Falling Prices, Downward Pressure (CPI, PPI, GDP Deflator Or Deflator, Commodities). Lower Prices. Deflationary Pressure, Lower Inflation Deflation -1 Deflation Excess liquidity 1 Excess Liquidity Enhanced liquidity 0.5 Ample or Enhanced Liquidity, Increased (Injected) Liquidity; Moderately Accommodative Liquidity, Generally Accommodative Liquidity, Generous, Persistently Accommodative Moderate liquidity 0 Lack of liquidity -0.5 Moderate, Reasonable Liquidity Shrinking liquidity -1 Liquidity Levels Shrink High level of employment 1 High Employment Rate, Low Unemployment Rate Increased employment levels 0.5 Moderate employment level 0 Stable Employment, Reasonable Employment Levels Lower employment levels -0.5 decline In Employment, Increase in Unemployment Very low level of employment -1 Insufficient Level of Liquidity Unemployment Pressure Eases, Overall Employment Level is Good, Employment Growth, Capacity Increases, Employment Status Index Improves and Increases Employment (Situation) is Expected to Improve High Pressure on Employment and Unemployment; Deteriorating Employment Situation (Not Optimistic); Unemployment Rate at Historically High (Continued ) PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 12 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking Table 1. (Continued) Phrase Type Subdivision Assignment value Real Estate Market Status Housing market overheating 1 Rising housing market 0.5 Detailed Expression Overheated, Surging, Persistently High, Rising Too Fast Growth in Commercial Property Sales, Rebound, Improvement, Rapid Sales Growth, Active Real Estate Market Transactions Home Sales Price Increases (Year-on-Year, Month-on-Month) Increase in Commercial Real Estate Loan Balances New Balance of Real Estate Development Loans Home Purchase Loans, Personal Housing Loans Added Increase in Real Estate Loans, Increase in The Proportion of Real Estate Development Investment (Year-on-Year, Month-on-Month) Stable housing market 0 Falling housing market -0.5 Stabilize Sales of Commercial Properties Fall, Real Estate Market Continues to Decline Home Sales Price Decline (Year-on-Year, Month-on-Month) Decrease in Commercial Real Estate Loan Balances Decrease in Balance of Real Estate Development Loans Decrease in Home Purchase Loans Lower Proportion of Real Estate Development Investment (Year-on-Year, Month-on-Month) Housing Market Recession -1 Depressed, Under Pressure, No Improvement, Continued Decline Note: Description of the assigned values. Drawing on Boschen and Mills [63] and Wang et al. [26], each of the above types of wording was divided into five magnitudes and assigned weights of 1, 0.5, 0, -0.5, and -1 according to wording strength and direction, respectively. https://doi.org/10.1371/journal.pone.0275110.t001 frequencies were weighted and summed. The details are as follows. Xn CIt ¼ i¼1 nobs ðxit Þ meannobs ðxi Þ ai std ðxi Þ ð21Þ where CIt is the central bank communication index in period t, which is the result of standardizing the effective wording xi, nobs (xit) is the word frequency of xi in period t of the Report, meannobs (xi) is the average word frequency of xi in each period of the Report, std (xi) is the standard deviation of the word frequency of xi in each period of the Report, and αi is the weight of effective wording xi, which is obtained by calculating the percentage of deviation. The final constructed central bank communication index CIt (cineit and ciwait are calculated separately, but the method is exactly the same) is proportional to the easing message of the central bank communication. The larger the index is, the more easing the central bank communication regulation is in that period. 4.2.2. Shadow banking indicators. Most of the literature selects its size indicator for the empirical study of Chinese shadow banking. The current methods used by scholars to measure the size of shadow banking mainly include macro estimation by the backward projection of shadow banking financing according to an estimated ratio of GDP or summing up the estimated size of each component of shadow banking, and the method of replacing the whole with the part by summing up the size of the main operations of undiscounted bank acceptance bills, entrusted loans, and trust loans [64, 65]. The macro estimation method has strong subjectivity and poor accuracy, and the estimation aggregation method has problems with double-counting, long intervals, and low data frequency. Therefore, combined with the comprehensive consideration of the research content and at the same time to avoid overestimating of the shadow PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 13 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking banking scale, we use the method of partial instead of whole. The method is adopted by most of the literature to calculate the growth rate of undiscounted bank acceptance bills, entrusted loans, and trust loan scale as shadow banking proxy variables (sbschr). 4.2.3. Bank risk-taking indicators. The current selection of bank risk-taking variables has two main perspectives: active and passive. First, the active risk-taking measures include the risk-weighted asset ratio [66, 67], nondeposit liabilities ratio [59, 68], expected default rate [28], and bank lending standards [11, 12, 61]. Second, the passive risk-taking measures are the nonperforming loan ratio [60, 66], Z-value [8, 35, 39, 69], and loan loss provisioning ratio. Based on previous experience and combined with the novel theory, we select the proportion of nondeposit liabilities (rndd) as a proxy variable for banks’ (active, liability-side) risk-taking based on a comprehensive consideration of multidimensional bank risk-taking. The nonperforming loan ratio (nlr), which can reflect banks’ passive, asset-side risk-taking, is taken as a robustness test indicator. 4.2.4. Bank-level factor indicators. Referring to existing studies [6, 68, 69], we select banks’ capital adequacy ratio (car) as a microlevel proxy variable. 4.2.5. Macroeconomic conditions and policy environment indicators. According to Maddaloni and Peydro [6] and Xu and Chen [69], the effect of the duration of the accommodative monetary policy variable (plmp) is incorporated by running an OLS regression of the annual benchmark interest rate on loans on the growth rate of the economy (gdp) and the growth rate of the price level (cpi) to obtain Taylor residuals (tr), with positive residuals representing a tight monetary policy environment (relatively high monetary policy rate), negative residuals representing an accommodative monetary policy environment (relatively low policy rate), and the duration of accommodative money (plmp) being the number of years in which the residuals are continuously negative. The data sources and descriptions of the main variables are shown in Table 2. The sample dataset is from 2009 to 2019. For the very few missing data points, the mean values of the adjacent two years were taken to check and fill. Then balanced panel data were generated and completed by STATA system operation. 4.3. Statistical characteristics of variables and data smoothness test 4.3.1. Descriptive statistics of each variable. Descriptive statistics for the main proxy variables are presented in Table 3. The mean value of the central bank communication index (cinei) is -0.080, with a maximum value of 6.690 and a minimum value of -6.090, indicating that the attitudes and intentions reflected in central bank communication vary considerably from period to period. The average growth rate of the shadow banking scale is -3.12%, the Table 2. Specific description of the main variables and data sources. Variable Name Specific instructions Data source Central Bank Communication Index (cinei, ciwai) Monetary Policy Implementation Report wording extraction, calculation construction Author measurement Shadow banking scale growth rate (sbschr) The sum of the growth rate of entrusted loans, trust loans and undiscounted bank acceptance bills WIND Percentage of non-deposit liabilities (rndd) Percentage of non-deposit liabilities = non-deposit liabilities/total assets WIND Non-performing loan ratio (nlr) Non-performing loan ratio = Non-performing loans/total loans WIND Bank Capital Adequacy Ratio (car) Capital adequacy ratio = net capital / risk-weighted assets WIND Duration of accommodative monetary Regressing interest rates on economic growth and cpi, the residuals of which are Taylor’s rule residuals, policy (plmp) using Taylor’s rule residuals less than zero for the duration Author measurement https://doi.org/10.1371/journal.pone.0275110.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 14 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking Table 3. Descriptive statistics of each variable. Variables N Mean Sd Max Min P50 P25 P75 cinei 385 -0.080 3.190 6.690 -6.090 -0.030 -2.430 1.820 ciwai 385 -0.950 2.300 2.450 -4.870 -0.830 -2.550 1.040 sbschr 385 -3.120 88.06 126.5 -196.0 -6.690 -47.26 63.45 rndd 385 23.78 10.42 49.90 0 23.45 16.70 31.58 nlr 385 0.520 3.880 0.160 1.190 0.860 1.520 car 385 2.440 40.30 8.330 12.97 11.87 14.10 plmp 396 1.338 4 0 1 0 2 1.230 13.14 1.182 https://doi.org/10.1371/journal.pone.0275110.t003 maximum value is 126.5%, and the minimum value is -196%, mainly because after the financial crisis in 2009, China introduced a series of new regulations and policies on shadow banking to control the scale of shadow banking. The guiding effect of preventing systemic risk is pronounced, and the growth rate of China’s shadow banking scale is on a downward trend after 2016. The banks’ risk-taking proxy variable nondeposit liability ratio (rndd) has a mean value of 23.78%, a maximum value of 49.9%, and a minimum value of 0. Meanwhile, the nonperforming loan ratio (nlr) has a mean value of 1.230%, a maximum value of 3.88%, and a minimum value of 0.16%, indicating that risk-taking varies widely across banks. The average value of the duration of accommodative monetary policy (plmp) is 1.182, with a maximum value of 4 and a minimum value of 0. This indicates that China’s central bank adopts a monetary policy with varying degrees of accommodative tightness in different periods of economic development, and the duration and the width of adjustment are larger. The capital adequacy ratio (car) has a mean value of 13.14%, a maximum value of 40.3%, and a minimum value of 8.33%, which are in line with reality. 4.3.2. Data stability test. To avoid spurious regression in the PVAR model, a unit root test of the variables is needed. The common LLC and IPS tests are selected here, and the results are presented in Table 4, where d_plmp is the unit root test of the variable plmp after the firstorder difference, and the P value are all significantly 0 at the 1% level. The P value of the unit root tests of the original data of the other variables all reject the original hypothesis of the existence of a unit root at the 1% level, indicating that each panel variable is smooth and satisfies the requirements of further tests. 5. Empirical tests and analysis of results 5.1. Central bank communication, shadow banking and bank risk-taking test results Prior to the PVAR model analysis, this study first determined the optimal lag order to be 1 based on the AIC, BIC and HQIC. Stability tests are then carried out to demonstrate that the Table 4. Results of the unit root test of variables. Variables LLC IPS T-value P-value Stability Statistic P-value Stability cinei -30.1755 0.0000 Stable -21.3200 0.0000 Stable ciwai -9.9520 0.0000 Stable -3.3533 0.0004 Stable sbschr -18.7281 0.0000 Stable -13.6004 0.0000 Stable rndd -8.4201 0.0000 Stable -2.5179 0.0059 Stable nlr -8.7730 0.0000 Stable -3.6866 0.0001 Stable car -7.6025 0.0000 Stable -3.5868 0.0002 Stable d_plmp -10.4802 0.0000 Stable -4.7454 0.0000 Stable https://doi.org/10.1371/journal.pone.0275110.t004 PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 15 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking system of variables constructed in this paper was stable. The correlation between the variables is again confirmed by Granger causality tests, which find that the central bank communication index (cinei), the growth rate of shadow banking size (sbschr) and the duration of accommodative monetary policy (d_plmp) are Granger causes of active bank risk-taking (rndd) but not the capital adequacy ratio (car). The central bank communication index (cinei) and the duration of accommodative monetary policy (d_plmp) are causally related to each other. The absence of Granger causality between the central bank communication index (cinei) and the size of shadow banking (sbschr) supports, to some extent, the theoretical model’s assumption that shadow banking information is relatively hidden and that it becomes more difficult for central banks to understand and grasp the underlying economic state. Considering that both are again closely related to active risk taking by banks (rndd) as previously confirmed, this further proves the significance and feasibility of this paper’s research. 5.1.1. GMM results for the PVAR model. To facilitate the distinction between subsequent empirical models, the main regression model is defined in this study as Model (1). The PVAR model uses the GMM approach to estimate the parameters, with all variables lagged by one period, and the estimation results are presented in Table 5. The lagged one-period domestic central bank communication index (cinei) is significantly positively correlated with banks’ active risk-taking (rndd) at the 10% level with a coefficient of 0.4175, indicating that the higher the degree of written communication from the central bank on easing policy, the greater the risk-taking of banks, thus confirming that Hypothesis 1 holds. Meanwhile, the one-period lagged growth rate of shadow banking size (sbschr) is negatively correlated with bank risk-taking (rndd) at the 5% significance level with a coefficient of -0.0054, which verifies Hypothesis 2 that shadow banking negatively affects bank risk-taking in the short run, but exactly what effect it will have in the long-run must be analyzed in conjunction with the subsequent impulse response plots. 5.1.2. Impulse response analysis. Fig 1 presents a plot of the effects of 200 Monte-Carlo shocks between the five variables of interest in this paper: duration of accommodative monetary policy, shadow banking, central bank communication index, bank capital adequacy, and bank risk-taking. The number of lags is 10, and the top of each subplot is labeled "impulse: response" (impulse variable: response variable). The solid line is the 50% quantile level, and the gray part is the confidence interval at the 95% level. Based on the impulse response diagram, the following specific analysis is made. There are existing tests of the monetary policy risk-taking channel in China (d_plmp— rndd). The first panel of the first row of Fig 1 shows that a one-unit shock to the duration of monetary policy (d_plmp) induces a 1.6902-unit isotropic change in bank risk-taking (rndd), Table 5. Model (1) PVAR model estimation results. d_plmp sbschr d_plmp(L1) -0.6540��� -10.9100 (0.0939) (13.7725) sbschr(L1) -0.0002 (0.0005) (0.0661) cinei(L1) -0.4081��� -5.1113 car(L1) car rndd 0.3329��� 1.6902��� (0.2548) (0.0917) (0.5491) 0.0011 -0.0003 -0.0054�� (0.0006) (0.0022) -0.0476 0.4175� (0.0013) 0.5493��� (0.0323) (5.5399) (0.0909) 0.1156 -60.9159�� -0.8929�� (0.0913) rndd(L1) -0.3384��� cinei 1.0000��� 0.1369��� (0.0174) (27.6741) (0.3845) -11.4864��� -0.3876��� (3.9454) (0.0590) (0.0438) 0.5209��� (0.1145) 0.0595��� (0.0193) (0.2266) -1.2436 (0.7554) 0.4175��� (0.1180) https://doi.org/10.1371/journal.pone.0275110.t005 PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 16 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking Fig 1. Model (1) 200 Monte-Carlo shock response graph. https://doi.org/10.1371/journal.pone.0275110.g001 which is lagged by one period and reaches a maximum in the first period, then turns weakly negative in the second period, but approaches zero and stabilizes in the third period and beyond. It is evident that an accommodative monetary policy environment will incentivize banks to increase their risk-taking. The bank risk-taking channel is actually more concerned with the impact of a sustained accommodative monetary policy environment rather than a short-term environment [5]. This point has long been easily overlooked, and this paper demonstrates that a channel for monetary policy risk-taking also exists in China from a perspective other than purely the policy interest rate or money supply [67, 69, 70]. There is evidence that accommodative monetary policy affects central bank communication and exchange (d_plmp—cinei). According to the second graph in the first row of Fig 1, it can be seen that a unit shock to the duration of easing monetary policy (d_plmp) can cause a positive response of approximately 1 unit to the central bank communication index (cinei) in the first period, and this positive relationship changes to a negative relationship in the second period, and stabilizes in the third and fourth periods close to 0. This shows that easing monetary policy positively affects the central bank’s communication on the easing economy and policy in the short term. The degree is pronounced, but with the growth of the duration of the easing monetary policy, the impact on the subsequent central bank communication index decreases or even becomes negative. With the increase in the implementation of the easing monetary policy time, the macroeconomic situation improves. Based on the principle of camera choice, central bank communication and communication will gradually become moderate, robust, or even tight communication. Hypothesis 1 is evidence that central bank communication and exchange on accommodative economic policies positively affect bank risk-taking when shadow banking exists (cinei— rndd). According to the third panel in the first row, a positive shock to the central bank’s written communication index (cinei) causes a positive response of 0.4175 units in bank risk-taking (rndd) in the first period, after which this positive relationship slowly weakens to a negative PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 17 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking response of approximately 0.2 units maximum in the second period, but then returns to a positive response in the third period, and maintains this stable positive relationship in the following periods until the eighth period when it gradually returns to near 0. This shows that the impact of central bank communication (cinei) on bank risk taking (rndd) is higher. In the short run, this effect is more volatile and more pronounced. Overall, a positive relationship can be maintained between the two. This is because central bank communication provides more information about the future, reduces uncertainty about policy and economic conditions, and increases the accuracy of commercial banks’ information forecasts. Central bank communication releases a sense of commitment from the central bank to the future. In the "odyssey" model of forwarding guidance (where central bank communication is a commitment), this feeling is amplified, and the effectiveness of central bank communication is enhanced [71]. Therefore, the increase in the strength and commitment of central bank communication to easing encourages banks to take higher risks; thus the empirical evidence supports Hypothesis 1. We combine the above two parts of the analysis. It can be seen that "monetary policy continues to be eased—central bank communication index increases—bank risk-taking increases". This suggests that the central bank communication and exchange mechanism of the bank risktaking channel exists in China. The easing monetary policy encourages banks to take higher risks by causing an increase in central bank communication. Hypothesis 2 shows that shadow banking in the context of central bank communication affects bank risk-taking (sbschr-rndd). In the short run, shadow banking (sbschr) favors lower bank risk-taking (rndd), but in the long run shadow banking (sbschr) weakly and positively affects bank risk-taking (rndd). Looking at the first panel of the second row of Fig 1, a one-unit positive shock to the growth rate of shadow banking size (sbschr) will cause a decline in bank risk-taking (rndd), which is lagged by one period and produces a maximum negative impact of approximately 0.0054 units after one year. Then this negative impact shrinks and becomes a small positive impact and remains stable in the fourth period and beyond. Obviously, this is in line with the characteristics of the operation mode of shadow banking in reality and the interdependent relationship with banks. To circumvent the regulation, commercial banks can use shadow banking innovation as an immediate response. It achieves off-balance-sheet funding through channel business, interbank business, and nested business. The asset and liability position of commercial banks can be improved. Especially in the short term, shadow banking can help commercial banks offset the impact of adverse monetary policy changes and reduce their risk-taking. However, in the long run, shadow banking without supervision often has unregulated operations and relatively higher risks in investment and financing. It is embedded in many financial intermediaries and financial instruments, making the entire system is relatively fragile. When the macro environment is good, the cash flow is stable, there are no special shocks, and the risks are not exposed, the impact on the risk-taking of commercial banks is small. However, in a special event, its apparent separation and close relationship with commercial banks makes it difficult to effectively separate risks and is highly contagious, which leads to an increase in risk-taking of commercial banks and an increase in the growth rate of shadow banking. There is a dynamic relationship between shadow banking and central bank communication. According to the second panel of the second row of Fig 1, a one-unit positive shock to the growth rate of shadow banking size (sbschr) causes an anisotropic response of 0.0011 units in the central bank communication index (cinei). It reaches a maximum of approximately 0.0025 units in the second period, finally declining gradually to approach zero in the eighth period and stabilizing. In general, to prevent the risk of the shadow banking system, the central bank’s communication and exchange of monetary policy should be tightened when shadow banks PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 18 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking expand. This deviation may be because the shadow banks that avoid regulation increase the difficulty and deviation of the central bank in grasping the financial system and the basic economic state. According to the third panel in the second row of Fig 1, a one-unit positive shock to the central bank communication index (cinei) causes a response of -5.1113 units in the growth rate of shadow banking size (sbschr) in the first period. This negative response turns positive in the second period and reaches a maximum in the third period before gradually returning to zero in the sixth period. The above two sections show a long and complex dynamic relationship between shadow banking and central bank communication. A bank’s capital adequacy negatively affects its risk-taking (car—rndd). According to the first panel in the third row of Fig 1, a one-unit shock to bank capital adequacy (car) causes a negative response in bank risk-taking (rndd). This negative relationship is lagged by one period, reaching a maximum in the third period and then gradually weakening, approaching zero and stabilizing in the seventh period. This is clearly in line with the majority of the literature on bank heterogeneity, which concludes that banks with higher capital adequacy ratios have lower levels of risk-taking. 5.1.3. Analysis of variance decomposition. According to the impulse response diagram, it is easy to see an up-and-down relationship between central bank communication, shadow banking, and bank risk-taking, which is not linear. The connection between the three is still somewhat complex. The specific variance decomposition results are shown in Table 6. The variance decomposition results in periods 9 and 10 are consistent, which means that the system is stable after nine prediction periods. Analyzing the variance decomposition results of banks’ risk-taking is the main concern of this paper. ① Banks’ risk volatility mainly originates from themselves, and although their contribution decreases from 64% in the first period to a stable 53.9% in the ninth period and beyond, their own contribution still accounts for the vast majority. ② The contribution of the positive effect of central bank communication on banks’ risk-taking increases from 7.67% in the first period to a maximum of 9.95% in the second period, then gradually to 9.25% in the ninth period and stabilizes. Yang and Zhou [72] argue that a variance decomposition contribution greater than 5% would indicate a causal relationship between the variables and is significantly valid, showing that the central bank communication index does significantly affect the risk-taking of banks, again proving Hypothesis 1 that central bank communication and communication positively affect the risk-taking of banks. ③ The contribution of shadow banking to banks’ risk-taking decreases from 27.1% in the first period to 24.4% in the second period relatively quickly, and then slowly decreases to 23% in the ninth period, which is the secondlargest contributing factor to banks’ risk-taking itself, indicating that the impact of shadow banking on banks’ risk-taking is not only great in the short term but also very significant in the long term. This is consistent with short-term banks tending to engage in risky businesses that avoid regulation with the help of shadow banking, which affects their risk-taking relatively quickly, while shadow banking, after long-term operation and fermentation, accumulates riskiness and vulnerability and is prone to backfire and contagion in the banking system in the long run, supporting Hypothesis 2. In an analysis of variance decomposition of central bank written communication (cinei), it is worth noting the contribution of its own (cinei) and the duration of accommodative monetary policy (d_plmp). ① The contribution of its own lagged one period decreases relatively quickly from 17.7% in the first period to 11.5% in the second period, to 10.3% in the fifth period and gradually stabilizes, while combined with the third graph in the third row of Fig 1. It shows that there is a strong correlation and succession between the adjacent written communication of the central bank before and after. The textual difference pairs are smaller, but the degree of influence on written communication decreases after two periods, which is consistent PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 19 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking Table 6. Model (1) variance decomposition results. Step Response Variable Impulse Variable Response Variable d plmp sbschr cinei car rndd Impulse Variable d plmp sbschr cinei car 1 d plmp 1 0 0 0 0 car 0.0424 0.0857 0.0191 0.853 rndd 0 2 d plmp 0.655 0.0164 0.0471 0.000970 0.281 car 0.175 0.0631 0.0138 0.712 0.0362 3 d plmp 0.480 0.0495 0.0750 0.00765 0.388 car 0.215 0.0492 0.0112 0.577 0.148 4 d plmp 0.448 0.0593 0.0732 0.0126 0.407 car 0.213 0.0485 0.0146 0.477 0.247 5 d plmp 0.426 0.0606 0.0705 0.0183 0.425 car 0.208 0.0533 0.0185 0.423 0.298 6 d plmp 0.413 0.0651 0.0722 0.0212 0.429 car 0.201 0.0571 0.0209 0.398 0.323 7 d plmp 0.408 0.0668 0.0726 0.0247 0.428 car 0.196 0.0605 0.0230 0.387 0.334 8 d plmp 0.407 0.0671 0.0726 0.0266 0.427 car 0.193 0.0624 0.0241 0.385 0.336 9 d plmp 0.407 0.0672 0.0726 0.0273 0.426 car 0.192 0.0632 0.0245 0.384 0.336 10 d plmp 0.407 0.0672 0.0725 0.0276 0.426 car 0.192 0.0634 0.0246 0.385 0.335 1 sbschr 0.157 0.843 0 0 0 rndd 3.18e-05 0.271 0.0767 0.0129 0.640 2 sbschr 0.116 0.742 0.00433 0.0764 0.0612 rndd 0.0140 0.244 0.0995 0.0710 0.572 3 sbschr 0.153 0.704 0.00477 0.0736 0.0646 rndd 0.0134 0.240 0.0941 0.0994 0.554 4 sbschr 0.149 0.681 0.00459 0.0711 0.0938 rndd 0.0159 0.238 0.0953 0.110 0.541 5 sbschr 0.149 0.667 0.00797 0.0697 0.106 rndd 0.0191 0.236 0.0945 0.114 0.536 6 sbschr 0.148 0.662 0.00840 0.0700 0.112 rndd 0.0219 0.234 0.0937 0.115 0.536 7 sbschr 0.147 0.659 0.00877 0.0706 0.114 rndd 0.0241 0.232 0.0930 0.114 0.537 8 sbschr 0.147 0.658 0.00902 0.0710 0.115 rndd 0.0250 0.231 0.0926 0.113 0.538 9 sbschr 0.147 0.658 0.00908 0.0714 0.115 rndd 0.0254 0.230 0.0925 0.113 0.539 10 sbschr 0.147 0.658 0.00909 0.0715 0.115 rndd 0.0255 0.230 0.0925 0.113 0.539 1 cinei 0.615 0.208 0.177 0 0 2 cinei 0.418 0.133 0.115 0.0471 0.287 3 cinei 0.360 0.129 0.119 0.0406 0.352 4 cinei 0.345 0.123 0.107 0.0401 0.385 5 cinei 0.326 0.121 0.103 0.0406 0.409 6 cinei 0.315 0.123 0.104 0.0426 0.415 7 cinei 0.311 0.124 0.103 0.0458 0.415 8 cinei 0.310 0.125 0.103 0.0476 0.415 9 cinei 0.309 0.125 0.103 0.0485 0.414 10 cinei 0.309 0.125 0.103 0.0489 0.414 https://doi.org/10.1371/journal.pone.0275110.t006 with the characteristics and patterns of the Monetary Policy Implementation Report issued by the People’s Bank of China. ② The contribution of the duration of accommodative monetary policy (d_plmp) to the central bank’s written communication index (cinei), although declining, has always occupied an important proportion, gradually declining from the initial 61.5% to 30.9% and stabilizing. This shows that the accommodative macroeconomic conditions and policy environment clearly influence the central bank’s communication and exchange, especially the short-term effect, which is extremely significant. The reality is clearly such that the Monetary Policy Implementation Report is indeed a summary of the monetary policy and macroeconomic conditions of the approaching period and a look at future policy and economic development prospects based on the recent domestic and foreign macro policy environment. The duration of the easing policy and economic conditions at the time of the Report’s writing plays an absolutely dominant position in influencing the content of the Report, which supports the first half of the central bank communication and exchange mechanism of the monetary policy bank risk-taking channel proposed by Blinder [30] and Borio and Zhu [5]; in other words, easing monetary policy influences central bank communication and exchange. PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 20 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking Fig 2. 200 Monte-Carlo impulse response plots for Model (2) under small and medium-sized joint-stock banks. https://doi.org/10.1371/journal.pone.0275110.g002 5.2. Robustness tests To ensure the robustness of Model (1), various empirical tests such as substituting bank risktaking variables, considering central bank communication on foreign policy and economic conditions and changing the order of the main variables, are carried out, and the PVAR2 method is used in these tests. Unlike the PVAR approach in the main regression, the first variable appearing in the impulse response plot under the PVAR2 approach is the response variable and the second variable is the shock variable. The robustness tests are all in general agreement with the main regression, and the results are not presented due to space constraints. Test results are available from the author on request. It is worth noting that the central bank foreign communication index (ciwai) negatively affects bank risk-taking (rndd), suggesting that bank risk-taking also responds to the Report’s communication and exchange on the macro environment in other countries, with a lower central bank foreign communication index (ciwai) representing a relatively poor foreign policy situation, in contrast to a potentially relatively accommodative domestic policy, which would clearly stimulate domestic commercial bank risk-taking, confirming Hypothesis 1 in reverse. 5.3. Test results and analysis based on the heterogeneity of banks The previous section has explained that banks may differ in their risk-taking capacity depending on their size and nature of ownership. Therefore, we divide banks into large state-owned banks (Bank of China, Agricultural Bank of China, Industrial and Commercial Bank of China, China Construction Bank, Bank of Communications, and Postal Savings Bank of China). The PVAR model for the sample of small and medium-sized joint-stock banks is denoted as Model (2). The PVAR model for the sample of large state-owned banks is denoted as Model (3) when the bank risk-taking proxy variable is the proportion of nondebt deposits (rndd). Due to space limitation, its unit root test, lag selection test, smoothness test, Granger causality test and variance decomposition analysis are not listed, and only the impulse response plots of the key parts are given (see Figs 2 and 3). It is easy to see that the impulse response plots of small and medium-sized joint-stock banks (Fig 2) are largely consistent with those of the full sample of banks (Fig 1), but the positive response of risk-taking to central bank communication is more pronounced for small and medium-sized joint-stock banks. According to the third panel in the first column of Fig 2, the coefficient of the positive effect of central bank communication on banks’ risk-taking in the Fig 3. 200 Monte-Carlo impulse response plots for Model (3) under large state-owned banks. https://doi.org/10.1371/journal.pone.0275110.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 21 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking first period is 0.5112 (according to the GMM results under the PVAR approach) greater than 0.4175 under the full sample in Model (1). However, the impulse responses of the large bank sample (Fig 3) GMM results under the PVAR method are not significant. The system is not stable and diverges significantly from the full sample of banks for the following possible reasons. ① Large state-owned banks have more channels to obtain time-sensitive information. The six major banks of China, Agriculture, Industry, Construction and Communications and Post and Reserve are naturally related to the central bank and have diversified. More rapid communication channels with the government and monetary authorities and access to information are less dependent on the central bank for communication than small and medium-sized joint-stock banks. ② Large state-owned banks mainly implement policies mainly and have lower information sensitivity. Since the founding of the country, large state-owned banks have been separated from the People’s Bank of China by their mother’s womb, shouldering the responsibility and cost of reform and transformation, their business behavior has been highly consistent with national policies and the relatively lagging Monetary Policy Implementation Report has had little impact on them. ③ The demonstration effect requires large state-owned banks to be transparent and legal. Due to the industry’s model responsibility and social responsibility, large state-owned banks are highly transparent and regulated, and their operations, including shadow banking, are relatively standardized and legal. Therefore, the impact of large state-owned banks’ risk-taking on their own risk-taking is inevitably smaller than the impact of nonstandardized and hidden shadow banking developed by small and medium-sized joint-stock banks. ④ The scale effect weakens the impact of shadow banking. Large state-owned banks are market leaders with absolute market positions and bargaining power, which weaken the influence of shadow banking on their bank risk-taking. The above supports Hypothesis 3 that the impact of central bank communication on bank risk-taking in the presence of shadow banking is more pronounced among small and medium-sized joint-stock banks than among large state-owned banks. 6. Further analysis Based on the above analysis, it is necessary to further explore the effect of the bank risk-taking channel under the growth rate of central bank written communication and shadow banking. To test what effect central bank communication can have on economic performance through the bank risk-taking channel in the context of the development of shadow banking, and to this end, we construct the corresponding PVAR empirical model in the following form: Xp Xit ¼ bi þ B X þ dt þ fi þ εit ð22Þ j¼1 j i;t j i 2 f1; 2; . . . . . . ; Ng; t 2 f1; 2; . . . . . . ; Tg where X_it is a 1 × m-dimensional vector of six variables at year t of the i-th bank, expanded as 0 1 EPEt B C B SB C B C t B C B CIt C C Xit ¼ B ð23Þ B C B BRTit C B C B C @ RISKit A EBit =SAit PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 22 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking Table 7. Description of the PVAR2 model for further testing. Model (4) Model (5) Model (6) Model (7) d_plmp d_plmp d_plmp d_plmp sbschr sbschr sbschr sbschr cinei cinei cinei cinei car car car car rndd nlr rndd nlr d_nni d_nni tacb tacb https://doi.org/10.1371/journal.pone.0275110.t007 Compared with Eq (19), a variable EBit/SAit is added here. EBit represents the profitability of banks, which is generally measured by the return on total assets or net assets, but the return on assets in the case of high leverage is volatile and easily distorted, which is not conducive to the study. Compared with the return on total assets, the ratio of noninterest income can better reflect the result of banks’ active risk-taking, so the ratio of noninterest income (d_nni) is selected as a proxy variable for bank profitability. The noninterest income share (nni) refers to the proportion of operating income other than the bank’s carry income to operating income. Noninterest income includes income from activities such as intermediation, consulting, and investment, where d_nni is the first-order difference of the variable nni, and its P value for both LLC and IPS unit root tests is significantly zero at the 1% level. SAit represents the asset size of banks, and the logarithm of the total asset size of banks (tacb) is used as a proxy variable. Specifically, it is divided into the four models in Table 7 below. Due to the space limitations, the Models’ (4)-(7) data smoothness test, determination of the optimal lag order, stability test, Granger causality test, and variance decomposition results in the further analysis are omitted, and only the impulse response analysis plots of the key parts of the four models are given. Figs 4 and 6 show the impulse responses of bank earnings and bank size to each factor, respectively, focusing on how shocks to bank risk-taking (rndd and nlr) cause changes in the response of the share of bank noninterest income (d_nni) and bank asset size (tacb). In Fig 4, both the ratio of nondeposit liabilities (rndd) and the nonperforming loan ratio (nlr) of banks hit the ratio of noninterest income (nni) of banks in the same direction in the same period, reaching a maximum in the first period and then gradually decreasing, returning to zero and remaining stable in the fourth and fifth periods. This shows that the increase of banks’ risk-taking causes banks’ profitability to improve, mainly due to the following reasons (see Fig 5). The higher the proportion of nondeposit liabilities of banks, the higher their funding costs tend to be, and the use of funds is bound to be more in pursuit of high returns. The Fig 4. Impulse response of bank noninterest income (d_nni) to each factor. https://doi.org/10.1371/journal.pone.0275110.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 23 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking Fig 5. Mechanisms by which bank risk-taking affects the share of non-interest income of banks. https://doi.org/10.1371/journal.pone.0275110.g005 high flexibility and liquidity of nondeposit liability funding sources make it easier to match the maturity and comprehensive management of assets and liabilities, which in turn makes the allocation of banks’ asset portfolios more reasonable, and increases the sources of noninterest income under the relatively stable situation of banks’ traditional carry income, thus causing the proportion of banks’ noninterest income to increase. An increase in the NPL ratio affects the bank’s interest income, which, other things being equal, decreases, resulting in a higher share of noninterest income. In Fig 6, the share of nondebt deposits (rndd) of banks positively shocks the total asset size of banks (tacb) in the immediate period and gradually decreases after reaching a maximum around the third period, but still maintains a positive relationship and remains stable. The bank nonperforming loan ratio (nlr) shocks the total bank asset size (tacb) negatively in the immediate period, but rises to a positive relationship around the second period and holds steady. The difference between the two models is related to banks’ active and passive risk-taking. Generally, a higher percentage of nondeposit liabilities of banks tends to mean that banks are more advanced in their operating concepts and methods and have a stronger ability to actively obtain funds (active liabilities), although the risk-taking is greater compared to deposit funds (passive liabilities), but the relatively stable deposit size does not easily restrict business development. It is conducive to the diversification of assets and expansion of scale, thus leading to the expansion of immediate bank size. The bank’s passive risk taking represented by the Fig 6. Impulse response of bank asset size (tacb) to each factor. https://doi.org/10.1371/journal.pone.0275110.g006 PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 24 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking non-performing loan rate is not quite the same. When the nonperforming loan rate rises, it may be due to certain problems in the bank’s management and risk control, or it may be due to the deterioration of the macroeconomic environment and the conditions of microenterprises and individuals, all of which will cause the bank to fail to recover the principal and interest on schedule. At the same time, it requires more special loss provisions, resulting in a reduction in the funds available to banks in the immediate period. Thus, in a short period of time, the bank’s passive risk taking (nlr) will cause a decline in the size of bank assets. However, when banks realize such problems, they will certainly update and improve their management and risk management levels, or macro policies will also be regulated to release more lower-cost liquidity, stimulating banks to use funds more prudently. Thus, banks’ passive risk-taking will still lead to asset size expansion in the long run. Therefore, the overall increase in bank risktaking (rndd and nlr) leads to the expansion of bank size, which supports Ayele’s [73] argument that bank risk-taking behavior is the Granger cause of bank size. 7. Conclusions and recommendations Central bank communication is widely used in the macroeconomic regulation of monetary policy. As an extremely important and frequently operated unconventional monetary policy tool, how central bank communication should reasonably serve the monetary policy transmission mechanism, act on the risk-taking of financial entities represented by commercial banks and enhance the effectiveness of monetary policy is an important challenge in building a leading modern central banking system and preventing and resolving systemic financial risks in China. At the same time, with the development of domestic financial innovation, shadow banking has flourished, effectively solving the problem of complex financing for some SMEs, but its scale expansion can also affect the operation of banks, especially in risk-taking. Therefore, this paper studies the relationship between central bank communication, shadow banking, and bank risk-taking based on the central bank’s most formal and authoritative Monetary Policy Implementation Report and further analyzes the possible economic consequences. The theoretical model and empirical tests find that ① the central bank’s written communication index positively affects bank risk-taking, verifying the existence of a central bank communication and exchange mechanism for the monetary policy bank risk-taking channel. ② The development of shadow banking may lead to a decline in banks’ risk-taking in the short term, but there is a stable positive correlation between the two in the long term. ③ The above findings are supported in robustness tests, and central bank communication on foreign policy and the economic environment is negatively related to bank risk-taking. ④ The above results are heterogeneous across ownership systems. In other words, they are more pronounced in small and medium-sized joint-stock banks and not significant in large state-owned banks. ⑤ Further research proves that bank risk-taking positively affects banks’ return profiles and asset size. In response to the above findings, the following policy recommendations are made. ① A good written communication strategy should constantly seek a more moderate frequency, neutral expression, and objective tone. In this way, banks and other market players can clearly understand the policy intentions of monetary authorities without creating overly optimistic expectations. Of course, this is difficult, and the monetary authorities also need to use other communication methods based on the nature of ownership (state-owned and joint-stock) and the size (large and small and medium-sized) of banks. For example, new social means such as central bank working papers, press conferences, speeches by central bank governors and officials, and official WeChat public numbers can soften the rigid expression of the Report and mitigate the lagging effect. This could provide the market with a clearer basis for decisionmaking. ② At the same time, the central bank should also pay attention to enhancing PLOS ONE | https://doi.org/10.1371/journal.pone.0275110 September 28, 2022 25 / 29 PLOS ONE Central bank communication, shadow banking, and bank risk-taking communication in the intricate global environment and major national policies to facilitate banks’ comprehensive understanding of the domestic and international situation and the attitude and position of the PBOC for scientific decision-making. ③ For shadow banking, while encouraging formal operations and legal and orderly development, it should also strengthen supervision and establish a targeted monitoring system, such as monitoring and guiding its fund flow areas. It should also try to let it serve small and microenterprises and high-tech enterprises that are difficult for formal finance to function in the short term. A perfect supervision system and transparent use of funds can effectively reduce the driving effect of shadow banking on banks’ risk-taking channels and facilitate risk prevention and resolution in the banking system. ④ In addition, the increase in banks’ risk-taking is not necessarily bad and may help banks increase the proportion of noninterest income and asset size. Banks should constantly strengthen their thinking on forward-looking central bank communication, global planning, and strategic layout, including shadow banking within a manageable degree of risk, to develop safety, quality, efficiency, and scale speed. Author Contributions Conceptualization: Jing Zhang, Shuai Chen, Honglei Liu. Data curation: Shuai Chen. Formal analysis: Jing Zhang, Shuai Chen, Honglei Liu. Funding acquisition: Jing Zhang. Methodology: Shuai Chen, Honglei Liu. Project administration: Jing Zhang. Resources: Jing Zhang. Software: Shuai Chen, Honglei Liu. Validation: Jing Zhang. 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W4232068554.txt

Qeios · Definition, February 2, 2020 Ope n Pe e r Re v ie w on Qe ios Failure of Cardiac Pacemaker to Pace National Cancer Institute Source National Cancer Institute. Failure of Cardiac Pacemaker to Pace. NCI T hesaurus. Code C99528. Failure to pace manifests as absence of pacemaker stimulation artifacts on electrocardiographic recordings despite rates below pacemaker programmed rate. Qeios ID: FAEHEB · https://doi.org/10.32388/FAEHEB 1/1 

W4393103813.txt

Jurnal Riset Rumpun Ilmu Teknik (JURRITEK) Vol. 2, No.2 Oktober 2023 e-ISSN: 2829-016X; p-ISSN: 2829-0178, Hal 11-20 DOI : https://doi.org/10.55606/jurritek.v2i1.1570 Analisis Waktu dan Biaya Berdasarkan Acuan Produktivitas Lapangan Menggunakan Metode Estimasi Biaya Aktual Rahmat Tri Prasetya Teknik Sipil Universitas Hasyim Asy’ari Tebuireng Jombang rahmattriprasetya01@gmail.com Totok Yulianto Teknik Sipil Universitas Hasyim Asy’ari Tebuireng Jombang totokyulianto@unhasy.ac.id Meriana Wahyu Nugroho Teknik Sipil Universitas Hasyim Asy’ari Tebuireng Jombang rian.sipilunhasy@gmail.com Titin Sundari Teknik Sipil Universitas Hasyim Asy’ari Tebuireng Jombang titinsundari1273@gmail.com Abstract. The improvement of the Cukir-Jombang road section requires heavy equipment. There is an increase in the road section at the STA. 0+000-STA. 0+936 and STA. 1+912 – STA. 2+790. In order to obtain cost and time efficiency, it is necessary to analyze the productivity of heavy equipment. This study uses the field productivity method with the aim of knowing the maximum productivity of heavy equipment and asphalt costs starting from STA 0+000 to completion. The results of this study obtained the rental time of heavy equipment for 8 hours with the number of each heavy equipment Asphalt Finisher 2 units, Dump Trucks 20 units, Tandem Roller 3 units, Pneumatic Tire Roller 2 units, Asphalt Sprayer 1 unit. Meanwhile, the cost of renting heavy equipment is IDR 4,180,000.00 for Asphalt Finisher, IDR 34,200,000 for Dump Trucks, IDR 5,700,000.00 for Tandem Rollers, IDR 3,800,000 for Pneumatic Tire Rollers, and IDR 532,000 for Asphalt Sprayers. Keywords: Work Volume, Production Capacity, Heavy Equipment Productivity, Estimated Actual Cost Abstrak. Dalam peningkatan ruas jalan Cukir-Jombang memerlukan alat berat. Peningkatan ruas jalan terdapat pada STA. 0+000-STA. 0+936 dan STA. 1+912 – STA. 2+790. Guna memperoleh efisiensi biaya dan waktu diperlukan analisa produktivitas alat berat. Penelitian ini menggunakan metode produktivitas lapangan dengan tujuan mengetahui maksimal produktivitas alat berat dan biaya pengaspalan mulai dari STA 0+000 sampai selesai. Hasil penelitian ini diperoleh waktu penyewaan alat berat selama 8 jam dengan jumlah masing-masing alat berat Asphalt Finisher 2 unit, Dump Truk 20 unit, Tandem Roller 3 unit, Pneumatic Tire Roller 2 unit, Asphalt Sprayer 1 unit. Sedangkan biaya penyewaan alat berat masing-masing sebesar, Asphalt Finisher Rp 4.180.000,00, Dump Truk Rp 34.200.000,00, Tandem Roller Rp 5.700.000,00, Pneumatic Tire Roller Rp 3.800.000,00, Asphalt Sprayer Rp 532.000,00. Kata kunci: Volume Pekerjaan, Kapasitas Produksi, Produktivitas Alat Berat, Estimasi Biaya Aktual LATAR BELAKANG Ruas jalan Cukir-Jombang merupakan salah satu ruas jalan utama wilayah selatan Kabupaten Jombang. Ruas jalan Cukir-Jombang juga menjadi ruas jalan strategis sebagai akses Received April 25, 2023; Revised Juni 27, 2023; Accepted Juli 24, 2023 *Rahmat Tri Prasetya, rahmattriprasetya01@gmail.com Analisis Waktu Dan Biaya Berdasarkan Acuan Produktivitas Lapangan Menggunakan Metode Estimasi Biaya Aktual penunjang Kawasan Wisata Religi Makam Gus Dur. Terdapatnya beberapa ruas jalan yang berlubang serta tidak adanya dinding penahan jalan membuat jalan tersebut memerlukan peningkatan jalan. Proyek peningkatan jalan berlokasi dijalan Mojowarno Cukir proyek ini dilaksanakan pada bulan juli sampai september proyek ini dimulai dari STA.0+000 yaitu dari Mojowarno ke arah Cukir dan titik terakhir selesai pekerjaan yaitu STA. 2+790, jalan ini menghubungkan Jombang kota ke Wonosalam untuk pekerjaannya tidak semua dilaksanakan peningkatan pengaspalan, proyek ini dikerjakan oleh perusahaan PT Sinar Abadi Citra Sarana. Guna mempermudah pembangunan peningkatan jalan dibutuhkan bantuan alat berat (Akbar et al., 2021). Produktivitas semua alat yang di pergunakan dalam pekerjaan jalan sangat berpengaruh terhadap proyek perkerasan jalan (Handayani & Akbar, 2020). Oleh karena itu, perlu diperlakukan analisa untuk memperoleh waktu dan biaya yang efisien sehingga proyek perkerasan jalan bisa selesai sesuai rencana. Dengan sempitnya ruas jalan yang menjadi titik pekerjaan, kepastian akan terjadi banyak hambatan yang akan terjadi di dalam penghamparan di lokasi pekerjaan. Sehingga penulis memutuskan untuk mengambil judul " Analisis Waktu dan Biaya berdasarkan Acuan Produktivitas lapangan Menggunakan Metode Estimasi Biaya Aktual (Studi Kasus: Peningkatan jalan Cukir - Mojowarno) ", agar mahasiswa mengetaui berapa maksimal produktivitas alat berat dan biaya pengaspalan mulai dari STA 0+000 sampai selesai. KAJIAN TEORITIS Perkerasan Jalan Terdapat beberapa klasifikasi perkerasan jalan, diantaranya: 1. Konstruksi perkerasan lentur (flexible pavement) Perkerasan dengan memakai aspal yang dijadikan sebagai pengikat disebut konstruksi perkerasan lentur (flexible pavement). Perkerasan in memiliki fungsi untuk menahan serta membagi beban lalu lintas menuju tanah dasar dari permukaan perkerasan. 2. Konstruksi perkerasan kaku (rigid pavement) Perkerasan dimana semen (portland cement) dijadikan sebagai bahan yang mengikat disebut konstruksi perkerasan kaku (rigid pavement). 3. 12 Konstruksi perkerasan komposit (composite pavement) JURRITEK - VOLUME 2, NO. 2, OKTOBER 2023 e-ISSN: 2829-016X; p-ISSN: 2829-0178, Hal 11-20 Kombinasi perkerasan dimana memadukan aspal serta semen (PC) untuk bahan yang mengikatnya disebut konstruksi perkerasan komposit (composite pavement). Analisis Biaya Alat Berat Perkerasan kaku tersusun oleh pelat beton semen portland serta lapisan dasar (mungkin iya/tidak) pada substrat dikenal dengan sebutan Perkerasan beton semen portland (Ardiansyah, 2020). Perkearasn dari pelat beton digunakan dengan atau tanpa tulangan dilihat pada desainnya. Beban pada area yang cukup luas didistribusikan oleh perkerasan kaku dengan modulusitas yang tinggi, Oleh karena itu pelat beton merupakan penyususn sebagian besar kapasitas struktur perkerasan. Produktivitas Alat Berat Berikut merupakan beberapa alat berat yang dipakai: 1. Asphalt Finisher Perhitungan produktivitas asphalt finisher dipakai rumus di bawah ini: (𝑃 = 𝑊𝑥𝑆𝑥𝐸𝑥𝑝) ........................................................................ Rumus 1 Keterangan: P = Produktivitas Asphalt Finisher (m3/jam) W = Lebar hamparan (m) S = Kecepatan gerak Asphalt Finisher (km/jam) E = Efisiensi kerja Asphalt Finisher p = Tebal lapisan (m) 2. Asphalt Sprayer Berikut data sesuai dengan spesifikasi teknis alat: Kapasitas tangki aspal, Cp = 850 liter, Tenaga penggerak, Pw = 4,0 HP. Kapasitas pompa aspal, pa = 55 liter/menit, Kapasitas produksi/jam, Q = pa x fa x 60 .................................. Rumus 2 Analisis Waktu Dan Biaya Berdasarkan Acuan Produktivitas Lapangan Menggunakan Metode Estimasi Biaya Aktual Kapasitas produksi/jam, Q = (pa x fa x 60)/lt ............................ Rumus 3 Dimana: pa ialah kapasitas pompa aspal, (0,55 liter / menit) Fa ialah faktor efisiensi alat (disebabkan faktor kesulitan serta keamanan kerja, maka diambil kondisi sedang) lt ialah pemakaian aspal (liter) per m² luas permukaan. (sebagai contoh 0,8 liter/m2) 60 merupakan konversi menit dari jam 3. Dump Truck Produktivitas Dump truck (Handayani & Akbar, 2020) adalah: P= 4. C × 60 × E Cmt ................................................................................. Rumus 4 P = produktifitas Dump truck (m3/jam) C = material di bucket Dump truck (ton) E = efisiensi kerja alat (meter) Cmt = waktu siklus (menit) Tandem Roller Perhitungan jumlah produksi per jam dari tundem roller compactor digunakan rumus (Primayandhi, 2018) sebagai berikut: P= (W X S X E)/N x P ............................................................... Rumus 2.5 Keterangan : 14 P = Produktivitas Tandem Roller (m3 / jam) W = Lebar Roller (meter) S = Kecepatan gerak Tandem Roller (Km/jam) E = Efisien kerja Tandem Roller (meter) N = Jumlah lewat bolak – balik (kali) p = Tebal lapisan Laston (meter) JURRITEK - VOLUME 2, NO. 2, OKTOBER 2023 e-ISSN: 2829-016X; p-ISSN: 2829-0178, Hal 11-20 5. Pneumatic Tyre Roller Perhitungan produktivitas (Sandi et al., 2019) yang dihasilkan dalam proses pemadatan aspal dengan memakai compactor untuk setiap tipe serta efisiensi waktu kerja alat dapat memakai rumus berikut : P= (W X S X E)/N x P ............................................................... Rumus 2.6 Keterangan : P = Produktivitas Pneumatic Tyre Roller(m3/ jam) W = Lebar Roller (meter) S = Kecepatan gerak Pneumatic Tyre Roller (Km/jam) E = Efisiensi kerja Pneumatic Tyre Roller (meter) N = Jumlah Lewat bolak – balik (kali) P = Tebal Lapisan Pemadatan (meter) METODE PENELITIAN Dalam penelitian ini memakai diskriptif kuantitatif. Guna pengumpulan data didapat melalui wawancara serta observasi. Berikut merupakan diagram alir penelitan guna mempermudah penelitian. Analisis Waktu Dan Biaya Berdasarkan Acuan Produktivitas Lapangan Menggunakan Metode Estimasi Biaya Aktual Mulai Rumusan Masalah Studi Literarur Pengumpulan Data Data Sekunder 1. RAB 2. TS 3. Spesifikasi Teknis Data Primer 1. Pengamatan 2. Wawancara Langsung Analisis Produktivitas Alat Berat Lapangan Analisis Uraian Teknis Penghamparan AC-BC dan AC-WC Analisis Waktu dan Biaya Pengerjaan Pembahasan Simpulan Selesai Gambar 1. Diagram alir penelitian 16 JURRITEK - VOLUME 2, NO. 2, OKTOBER 2023 e-ISSN: 2829-016X; p-ISSN: 2829-0178, Hal 11-20 HASIL DAN PEMBAHASAN 1. Perhitungan Volume Aspal Cair Berikut perhitungan volume pekerjaan penghamparan aspal cair Panjang jalan = 936 m Lebar jalan =6m Volume penghamparan aspal cair = 936m x 6m = 5.616 m2 Berikut merupakan tabel hasil perhitungan luas permukaan penghamparan aspal cair. Tabel 1 Volume Penghamparan Cair (Olah Data, 2021) Volume Aspal Cair Nama Ruas Segmen 1 Segmen 2 936,00 4,50 6,00 0,06 Kebutuhan aspal per m2 (liter/m2) 0,80 878,00 4,50 6,00 0,06 0,80 Panjang STA (m) Tahap 1 Lebar Awal (m) Lebar rencana (m) Tebal (m) TOTAL VOLUME AC – BC (m2) Segmen 1 Segmen 2 Tahap 2 5.616,00 5.268,00 10.884,00 936,00 4,50 6,00 0,04 0,80 5.616,00 878,00 4,50 6,00 0,04 0,80 5.268,00 TOTAL VOLUME AC – WC (m2) 2. Volume (m2) 10.884,00 Perhitungan Volume Asphal Hotmix Berikut perhitungan volume pekerjaan penghamparan AC-WC Panjang jalan = 878 m Lebar jalan =6m Tebal lapisan hotmix = 0.04 m Berat jenis Aspal = 2,28 ton/m3 Volume aspal yang akan di hamparkan = 878 m x 6 m x 0,04 m x 2,28 ton/m3 = 480,44 ton Berikut merupakan tabel hasil perhitungan penghamparan asphalt AC-BC dan asphalt ACWC. Analisis Waktu Dan Biaya Berdasarkan Acuan Produktivitas Lapangan Menggunakan Metode Estimasi Biaya Aktual Tabel 2 Volume Penghamparan Ashpalt AC-BC & AC-WC (Olah Data, 2021) Volume Aspal Nama Ruas Panjang STA (m) Segmen 1 Segmen 2 Tahap 1 Lebar Awal (m) Lebar rencana (m) Tebal (m) Berat Jenis Aspal (ton/m3) 936,00 4,50 6,00 0,06 2,30 775,01 878,00 4,50 6,00 0,06 2,30 726,98 TOTAL VOLUME AC – BC (ton) Segmen 1 Segmen 2 Tahap 2 1.501,99 936,00 4,50 6,00 0,04 2,28 512,18 878,00 4,50 6,00 0,04 2,28 480,44 TOTAL VOLUME AC – WC (ton) 3. Volume (ton) 992,62 Analisis Produktivitas Alat Berat 3.1. Produktivitas Asphalt Finisher 𝑄 = 𝑉 × 𝑏 × 60 × 𝐹𝑎 × 𝑡 × 𝐷1 = 207,25 𝑡𝑜𝑛/𝑗𝑎𝑚 3.2. Produktivitas Asphalt Sprayer 𝑄= 𝑝𝑎 × 𝐹𝑎 × 60 = 3.423,75 /𝑗𝑎𝑚 𝑙𝑡 3.3. Produktivitas Dump Truk 𝑄= 𝑉 × 𝐹𝑎 × 60 × 𝐷1 = 16,14 𝑡𝑜𝑛/𝑗𝑎𝑚 𝑇𝑠2 3.4. Produktivitas Tandem Roller 𝑄= (𝑣 × 1000) × (𝑁(𝑏 − 𝑏𝑜) + 𝑏𝑜) × 𝑡 × 𝐹𝑎 × 𝐷1 = 166,67 𝑡𝑜𝑛/𝑗𝑎𝑚 𝑛×𝑁 3.5. Produktivitas Pneumatic Tyre Roller 𝑄= 4. (𝑣 × 1000) × (𝑁 (𝑏 − 𝑏𝑜) + 𝑏𝑜) × 𝑡 × 𝐹𝑎 × 𝐷1 = 177,37 𝑡𝑜𝑛/𝑗𝑎𝑚 𝑛×𝑁 Analisa Waktu dan Biaya Berikut merupakan tabel hasil perhitungan kebutuhan alat berat pada pekerjaan asphalt AC-BC dan AC-WC. 18 JURRITEK - VOLUME 2, NO. 2, OKTOBER 2023 e-ISSN: 2829-016X; p-ISSN: 2829-0178, Hal 11-20 Tabel 3 Kebutuhan Alat Berat No. 1 2 3 4 5 Alat Berat Aspalt Finisher Dump Truk 6m3 Tandem Roller Pneumatic tire Roller Aspalt Sprayer Volume Aspal (per ton) Produktivitas Kebutuhan Alat Berat Jumlah Kebutuhan Alat Berat Durasi Sewa Selama 8 jam Jumlah Kebutuhan Alat Berat Dibulatkan AC-BC AC-WC AC-BC AC-WC ACBC ACWC 1.501,99 992,62 207,25 171,06 8,00 6,00 14,00 8 2,00 1.501,99 992,62 16,14 15,98 94,00 63,00 157,00 8 20,00 1.501,99 992,62 116,67 96,30 13,00 11,00 24,00 8 3,00 1.501,99 992,62 177,37 146,40 9,00 7,00 16,00 8 2,00 1.501,99 992,62 3.423,75 3.423,75 4,00 4,00 8,00 8 1,00 Diketahui kubutuhan jumlah alat berat untuk asphalt finisher ialah 2 unit, diperoleh dari perhitungan volume 1.501,99 ton dibagi produktivitas untuk AC-BC 207,25 ton per jam sehingga diperoleh 8 jam. Dan seterusnya untuk asphalt finisher AC-WC 6 jam sehingga total waktu 14 jam untuk alat asphalt finisher. Kebutuhan asphalt finisher sebanyak 2 unit karena kombinasi seluruh alat apabila dipakai selama 8 jam. Demikian seterusnya untuk perhitungan alat berat lainnya. Dalam perhitungan biaya alat berat dapat dilihat seperti tabel dibawah. Tabel 4 Biaya Alat Berat No. 1 2 3 4 5 Alat Berat Aspalt Finisher Dump Truk 6m3 Tandem Roller Pneumatic tire Roller Aspalt Sprayer Jumlah jam penyewaan Jumlah Waktu Penyewaan (jam) Jumlah Kombinasi Alat Dibulatkan Biaya Sewa (per jam) Biaya Sewa Alat 14,00 8 1,75 2,00 261.250,00 4.180.000,00 157,00 8 19,63 20,00 213.750,00 34.200.000,00 24,00 8 3,00 3,00 237.500,00 5.700.000,00 16,00 8 2,00 2,00 237.500,00 3.800.000,00 8,00 8 1,00 1,00 66.500,00 532.000,00 Diketahui besaran biaya sewa asphalt finisher ialah Rp 4.180.000,00 diperoleh dari jumlah alat berat yang dipakai 2 unit dikali jam pemakaian (8 jam) dikali biaya sewa perjam sebesar Rp 261.250,00. Demikian seterusnya diperoleh seperti perhitungan di atas. Analisis Waktu Dan Biaya Berdasarkan Acuan Produktivitas Lapangan Menggunakan Metode Estimasi Biaya Aktual KESIMPULAN DAN SARAN Dalam penelitian ini dapat diambil kesimpulan sebagai berikut: 1. Volume pekerjaan pengaspalan pada STA. 0+000 – STA. 0+936 ialah 775,01 ton untuk AC-BC dan 512,18 ton untuk AC-WC. Sedangkan pada STA. 2+790 – STA. 1+912 adalah 726,98 ton untuk AC-BC dan 480,44 ton untuk AC-WC. 2. Produktivitas dalam penghamparan aspal AC-BC untuk Asphalt Finisher 207,25 ton/jam, Dump Truk 16,14 ton/jam, Tandem Roller 116,67 ton/jam, Pneumatic Tire Roller 177,37 ton/jam, Asphalt Sprayer 3423,75 m2/jam. Sedangkan produktivitas dalam penghamparan aspal AC-WC untuk Asphalt Finisher 171,06 ton/jam, Dump Truk 15,98 ton/jam, Tandem Roller 96,30 ton/jam, Pneumatic Tire Roller 146,40 ton/jam, Asphalt Sprayer 3423,75 m2/jam. 3. Dari hasil penelitian diperoleh analisa waktu penyewaan alat berat selama 8 jam dengan jumlah masing-masing alat berat Asphalt Finisher 2 unit, Dump Truk 20 unit, Tandem Roller 3 unit, Pneumatic Tire Roller 2 unit, Asphalt Sprayer 1 unit. Sedangkan biaya penyewaan alat berat masing-masing sebesar, Asphalt Finisher Rp 4.180.000,00, Dump Truk Rp 34.200.000,00, Tandem Roller Rp 5.700.000,00, Pneumatic Tire Roller Rp 3.800.000,00, Asphalt Sprayer Rp 532.000,00. DAFTAR REFERENSI Akbar, F., Yamali, F. R., & Dwiretnani, A. (2021). Analisa Pengunaan dan Produktivitas Alat Berat pada Kegiatan Peningkatan Ruas Jalan Simpang Pauh – Air Hitam Provinsi Jambi. Jurnal Talenta Sipil, 4(2), 114. https://doi.org/10.33087/talentasipil.v4i2.57 Ardiansyah, R. (2020). Analisis Perencanaan Tebal Perkerasan Kaku Lajur Pengganti pada Proyek Pembangunan Jalan Tol Jakarta-Cikampek II Elevated. 05(01), 17–30. https://doi.org/10.29244/jsil.5.1.17-30 Handayani, E., & Akbar, F. (2020). Kajian Efisiensi Produktifitas Alat Berat pada Proyek Jalan (Studi kasus: Ruas Jalan Mendalo Darat (Sp.Tiga)-Bts.Kota Jambi). Jurnal Civronlit Unbari, 5(1), 16. https://doi.org/10.33087/civronlit.v5i1.63 Primayandhi, R. (2018). Studi Perhitungan Kebutuhan Alat Berat dan Biaya Lapis Pondasi Aggregat Kelas A Pada Jalan Sepunggur-Gunung Tinggi Kab. Tanah Bumbu. Jurnal Rekayasa Sipil, 2(1), 47–55. Sandi, F., Khamdari, E., & Pramono, E. (2019). Analisa Perbandingan Koefisien Harga Satuan Pekerjaan Pelapisan Ulang AC-WC (Studi Kasus : Ruas Jalan Tol Jakarta – Bogor – Ciawi Dan Ruas Jalan Tol Jakarta – Cikampek). Seminar Nasional Teknik Sipil Politeknik Negeri Jakarta. 20 JURRITEK - VOLUME 2, NO. 2, OKTOBER 2023 

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Order Code 95-36 Updated February 16, 2007 The Advanced Technology Program Wendy H. Schacht Specialist in Science and Technology Resources, Science, and Industry Division Summary The Advanced Technology Program (ATP) was created by P.L. 100-418, the Omnibus Trade and Competitiveness Act of 1988, to encourage public-private cooperation in the development of pre-competitive technologies with broad application across industries. Administered by the National Institute of Standards and Technology (NIST), a laboratory of the Department of Commerce, this activity has been targeted for elimination as a means to cut federal spending. Since FY2000, the original Housepassed appropriation bills have not included funding for ATP. Many of the Administration’s budget requests have proposed termination of the program. However, ATP continues to be supported, although at levels below that achieved in FY1995 when the activity was expanded significantly. The FY2005 appropriations were 20% lower than the earlier fiscal year. For FY2006, both the Administration’s budget proposal and the original House-passed version of the appropriations bill, contained no funding for ATP although P.L. 109-108, the final FY2006 appropriation legislation, financed the program at $79 million, 42% below the previous fiscal year (after a mandated rescission). The Administration’s FY2007 budget request again did not include funding for ATP, nor did the appropriation bill passed by the House and reported by the Senate Committee on Appropriations in the previous Congress. While no final FY2007 appropriations legislation was enacted during the 109th Congress, a series of continuing resolutions financed ATP at FY2006 levels through February 15, 2007. In the current Congress, P.L. 110-5 provides the program $79 million for FY2007. The President’s FY2008 budget request contains no funding for ATP. This report will be updated as events warrant. Program Rationale Title V of the Omnibus Trade and Competitiveness Act (P.L. 100-418) established the Advanced Technology Program (ATP). This effort grew out of concerns over the competitiveness of American companies in the global marketplace. While numerous factors affect the rate of technical progress in an economy, what was seen as critical is how quickly and successfully science and technology are transformed into new or better CRS-2 products, processes, or services. The commercialization and diffusion of goods and services stood out as significant problems in the ability of U.S. industries to compete. Underlying the structure of ATP is an effort to foster cooperation among government, industry, and academia to facilitate the generation of new technologies for the commercial market. While opponents argue that joint ventures stifle competition, proponents assert that they are designed to accommodate the strengths and responsibilities of various sectors. Collaborative projects attempt to utilize and integrate what the participants do best and to direct R&D activities toward the goal of meeting marketplace demands. Joint endeavors are seen as reducing risks and costs while permitting work that crosses traditional boundaries of expertise and experience. Program Operation The Advanced Technology Program was designed “to serve as a focal point for cooperation between the public and private sectors in the development of industrial technology” and to help solve “problems of concern to large segments of an industry,” as noted in the Conference Report to accompany the bill. Placed within the National Institute of Standards and Technology (NIST), in recognition of the laboratory’s ongoing relationship with industry, ATP provides seed funding to single companies or to industryled consortia of universities, businesses, and/or government laboratories for development of generic (broad-based), pre-competitive technologies that have many applications across industries. Awards, based on technical and business merit, are for high-risk work past the basic research stage but not yet ready for commercialization. Market potential is an important consideration in project selection. Scientific and technical review generally is performed by federal and academic experts. Business plan assessments are made by individuals from the private sector. Awards are for either product or process (manufacturing) technology development. Individual firms are restricted to funding of $2 million over three years. Money is to be used only for direct R&D costs. Large firms must provide at least 60% of total (direct and indirect) projects costs; small and medium-sized companies are not required to cost-share direct costs. Joint ventures may receive up to five years of financing for any amount limited only by availability. In such cases, the private sector must provide more than 50% of funding. While universities and federal laboratories can participate in collaborative work, the grant from ATP is made solely to companies. P.L. 102-245 modified the original law and required that the recipient of an ATP award be a firm that is U.S.-owned (“a company that has a majority ownership or control by individuals who are citizens of the United States”) or a business that is incorporated in the United States and has a parent company established in a country that affords American firms reciprocal opportunities. According to NIST, through the end of 2004 (when the last new awards were made), 768 projects were funded, of which about 30% were joint ventures. Approximately $2.3 billion in federal funds have been matched by $2.1 billion from the private sector. Small businesses or cooperative efforts led by such firms make up 65% of the awardees. The first four competitions (ending August 1994) were general in nature. However, in 1995 NIST restructured part of ATP to focus on various groups of projects in “well-defined” programmatic areas designed for long-range support. These were selected in conjunction with industry. Since FY1999 one competition has been held in all areas of technology. CRS-3 In its first year, FY1991, ATP was funded at $36 million. Appropriations increased to $48 million in FY1992, $67.9 million in FY1993, and $199.5 million in FY1994. For FY1995, financial support expanded significantly to $431 million. However, P.L. 104-6 rescinded $90 million of this amount. Funding for FY1996 was $221 million with a small increase to $225 million in FY1997 but reduced to $218 million by P.L. 105-18. For FY1998, P.L. 105-119 appropriated $192.5 million. P.L. 105-277 provided $197.5 million in FY1999 support for ATP, 3% above the previous year. This figure reflected a $6 million rescission contained in the same law that accounts for “deobligated” funds resulting from early termination of certain projects. In FY2000, the appropriations bill that originally passed the House included no funding for ATP, since, as stated in the accompanying report, “the program has not produced a body of evidence to overcome those fundamental questions about whether the program should exist in the first place.” Yet, P.L. 106-113 did finance ATP at $142.6 million, 28% less than FY1999. In FY2001, while the original House-passed appropriations bill did not fund the program, P.L. 106553 provided $145.7 million, 2% above the previous year’s financing. The Bush Administration’s FY2002 budget proposed suspension of new ATP projects pending a program evaluation, although $13 million was to be provided for ongoing financial commitments. Again, the initial appropriations bill passed by the House terminated ATP. The final legislation, P.L. 107-77, financed the effort at $184.5 million, almost 27% more than FY2001. The following year, the President requested $107.9 million in FY2003 funding for ATP. Several Continuing Resolutions financed the program until the 108th Congress enacted P.L. 108-7 which funded ATP at $178.8 million (after a 0.65% rescission), 3% below the earlier fiscal year. The Administration’s FY2004 budget requested $27 million for ATP (an 85% decrease) to cover on-going commitments; no new projects would be financed. The original House-passed appropriations bill provided no support for the program. However, P.L. 108-199 funded ATP at $170.5 million after mandated rescissions, 4.5% below FY2003 support. The next year, while the FY2005 budget proposal and the original appropriations legislation passed by the House did not include funding for ATP, P.L. 108447 provided the program with $136.5 million (after mandated rescissions), a decrease of 20% from the previous fiscal year. Both the President’s FY2006 budget and the original House-passed version of H.R. 2862, the FY2006 Science, State, Justice, and Commerce appropriations bill, again did not provide any support for ATP. The final FY2006 appropriation for ATP in P.L. 109108 was $79 million, 42% below FY2005 funding (after mandated rescissions). For FY2007, the Administration’s budget did not include funding for ATP nor did the appropriations bill initially passed by the House and reported from the Senate Committee on Appropriations during the 109th Congress. While no relevant FY2007 appropriations legislation was enacted in the previous Congress, ATP was financed at FY2006 levels through February 15, 2007 by a series of continuing resolutions. P.L. 1105 provides $79 million in FY2007 funding for the program, the same amount as FY2006. The President’s FY2008 budget request contains no support for ATP. Results CRS-4 NIST has undertaken numerous studies of ATP; the General Accounting Office (GAO, now the Government Accountability Office) has also studied the program. In its first evaluation (1994), NIST concluded the program had stimulated research that would not have been done without the federal support; that R&D cycles within companies have been abbreviated; and that “valuable business alliances” had been created.1 However, in a May 1995 report, GAO argued that these conclusions can not be adequately substantiated by the information provided in the NIST study on which they are based.2 Acknowledging that it was too early to determine the long-term impact of ATP, the GAO report stated that some of the indicators NIST utilized “may create false expectations of the program’s economic success.” NIST vigorously defended its methodology. Additional studies funded by NIST found that ATP shortened R&D cycles by half and accelerated technological progress within the firm; stimulated productive collaborative activities among companies and between firms and universities; facilitated commercialization; and increased private sector investment in high risk technology development.3 An April 2000 progress report reinforced these earlier findings.4 This study indicated that “participants in 261 projects have identified more than 1,200 different applications (or uses) of the technologies under development,” and that the majority of these are new solutions to market needs or improvements in existing products or processes. Product cycles are being reduced, and while 24% of respondents said that they would not have undertaken the project without ATP funding, most others noted that the R&D would have been significantly slower without such support. NIST found that “organizations are pursuing different R&D than they would have undertaken without ATP funding,” and that this work is more technically advanced and risky. The ATP financing also stimulated additional private sector money in these technical areas than otherwise would be the case. Over half of the companies are now able to make a new or improved product. According to March 9, 2000, testimony by Raymond Kammer, then director of NIST, approximately 120 new technologies have been commercialized. The concern over whether ATP supports projects that could reasonably attract private sector investment has been an issue throughout the life of the program. In a report examining award winners and “near winners” during the first four years of ATP, GAO found the program funded both projects that would not have progressed without this 1 National Institute of Standards and Technology, Setting Priorities and Measuring Results at the National Institute of Standards and Technology, January 31, 1994. 2 General Accounting Office, Performance Measurement, Efforts to Evaluate the Advanced Technology Program, GAO/RCED-95-68, May 1995. 3 Silber and Associates, Company Opinion about the ATP and Its Early Effects, January 30, 1995; Acceleration of Technology Development by the Advanced Technology Program: The Experience of 28 Projects Funded in 1991, by Frances Jean Laidlaw, for the National Institute of Standards and Technology, Economic Assessment Office, October 23, 1997; National Institute of Standards and Technology, Advanced Technology Program: Development, Commercialization, and Diffusion of Enabling Technologies, by Jeanne W. Powell, December 1997; National Institute of Standards and Technology, Advanced Technology Program Performance of Completed Projects, Status Report Number 1, by William F. Long, March 1999. 4 National Institute of Standards and Technology, Development, Commercialization, and Diffusion of Enabling Technologies: Progress Report, by Jeanne W. Powell and Karen L. Lellock, April 2000. CRS-5 federal support and those that would have been financed by the private sector.5 Half of the awardees stated that they would have continued without ATP financing. Of the “near winners,” 50% pursued their efforts in the absence of federal money but took longer to achieve their goals. According to GAO, while 63% of the applicants did not look elsewhere for funds, about half of the applicants who did “were told by prospective funders that their projects were either too risky or ‘precompetitive’ — characteristics that fulfill the aims of ATP funding.” Respondents also noted that the program facilitated development of joint ventures to pursue ATP activities. A study undertaken by the American Enterprise Institute concluded that ATP “has had only limited success” in choosing projects that could not raise private sector funds. According to the authors, this has occurred because companies are not interested in pursuing R&D that fails to complement work performed for profit. In addition, the ATP selection criteria focus on commercial sales and job creation, not on projects for which there are “broad social benefits” and insufficient private investment. An April 2000 report by GAO, reinforced by May 26, 2005 testimony, noted that “two inherent factors in ATP’s current award selection process — the need to guard against conflicts of interest and the need to protect proprietary information — make it unlikely that ATP can avoid funding research already being pursued by the private sector in the same time period.”6 Issues and Observations There have been efforts in the past several years to terminate the Advanced Technology Program. These actions, along with additional attempts to withdraw government support for other technology development efforts, appear to reflect a philosophy that eschews direct federal financing of private sector R&D efforts aimed at the commercialization of new technologies and production processes. Such activities are seen by opponents as “industrial policy,” the means by which government rather than the marketplace “picks winners and losers.” Instead, measures that would occasion a better investment environment for industry to expand their innovation-related efforts would, proponents argue, be preferable to government funding. This signals a change from the existing varied approach to facilitating technological advancement. Legislative initiatives have resulted in a body of laws, programs, and policies that involve both indirect and direct measures to stimulate technology advancement in the private sector. Indirect incentives include a research and experimentation tax credit; changes to the antitrust laws to encourage collaborative R&D and cooperative manufacturing ventures; alterations of patent ownership policies to facilitate government-industry-university interaction; and practices to promote technology transfer. Direct measures involve, among other things, federal funding for ATP and the Small Business Innovation Research Program. These cost-shared programs have been supported, in part, because of their potential contribution to the country’s national or economic security. 5 General Accounting Office, Measuring Performance: The Advanced Technology Program and Private-Sector Funding, GAO/RCED-96-47, Jan. 1996. 6 General Accounting Office, Advanced Technology Program: Inherent Factors in Selection Process Could Limit Identification of Similar Research, GAO/RCED-00-114, April 2000, 5. CRS-6 The mix of approaches was developed with bipartisan support in Congress. Under former President Reagan, public-private cooperation in research and development was promoted by the executive branch. The George H. Bush Administration adopted a policy in which the government’s role was “to support the development of generic or enabling technologies at the pre-competitive stage of R&D.” The Clinton Administration expanded this concept to include additional direct federal funding to achieve increased commercialization of the results of R&D. ATP reflects such ideas; thus discussions over its proposed elimination have called into question many of the underlying assumptions shaping the environment within which industry works toward technological advancement. Proposals to terminate or severely limit ATP have renewed the debate over the role of the federal government in promoting commercial technology development. In arguing for less direct federal involvement, advocates believe that the market is superior to government in deciding technologies worthy of investment. Mechanisms that enhance the market’s opportunities and abilities to make such choices are preferred. It is suggested that agency discretion in selecting one technology over another can lead to political intrusion and industry dependency. On the other hand, supporters of direct methods argue that it is important to focus on those technologies that have the greatest promise as determined by industry and supported by matching funds from the private sector. They assert that the government can serve as a catalyst for cooperation. Technological progress is important to the nation because of its contribution to economic growth and a high standard of living. How best to achieve this continues to be debated. Critics view ATP as a means for a federal agency to select commercial firms and/or technologies for support. They maintain that the absence of market-generated decisions will result in technologies that can not be utilized productively by participating companies. Such a program encourages selection of well-written proposals rather than assistance for truly important technologies. However, proponents stress that ATP is market driven and that the technical areas for investment have been developed in conjunction with industry. In addition, companies have to put up significant amounts of funding and survive a rigorous business review; procedures that make the Advanced Technology Program different from other federal efforts. Perhaps most crucial to the debate is the way cooperative R&D is viewed. Today, American companies appear to be more competitive in the global marketplace than when the 1988 Act was being considered. While there are many factors contributing to this improving situation, proponents of joint R&D efforts, such as ATP, point to the benefits derived from increased technical collaboration and the development and application of the resulting new technologies and production processes. Questions remain whether direct federal funding for such programs is the most effective or efficient means to secure these outcomes. Is the approach embodied in ATP the preferable one, or could other mechanisms such as permanent tax credits for R&D, changes in capital gains treatment, and/or liability and regulatory reform be more effective? 

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Selected Neurophysiological, Psychological, and Behavioral Influences on Subjective Sleep Quality in Nurses: A Structure Equation Model Min-Huey Chung1*, Wen-I Liu2, Hui-Ling Lee3, Nanly Hsu4 1 Graduate Institute of Nursing, College of Nursing, Taipei Medical University, Taipei, Taiwan, 2 School of Nursing, National Taipei University of Nursing and Health Sciences, Taipei, Taiwan, 3 Department of Nursing, Kang-Ning Junior College of Medical Care and Management, Taipei, Taiwan, 4 Nursing Department, Yuanpei University, Hsinchu, Taiwan Abstract Few studies have examined relationships among neurophysiological, psychological, and behavioral factors with regard to their effects on sleep quality. We used a structure equation model to investigate behavioral and psychological factors that influence neurophysiological regulation of sleep in shift workers. Using a cross-sectional study design, we tested the model with a sample of 338 female nurses working rotating shifts at an urban regional hospital. The Morningness-Eveningness Questionnaire (MEQ) and short-form Menstrual Distress Questionnaire (MDQ) were used to measure neurophysiological factors involved in morningness-eveningness and menstrual distress. The Sleep Hygiene Awareness and Practice Scale (SHAPS) and Profile of Mood States Short Form (POMS-SF) were completed to measure behavioral factors of sleep hygiene practices and psychological factors of mood states. In addition, the Pittsburgh Sleep Quality Index (PSQI) measured participant’s self-reported sleep quality. The results revealed that sleep hygiene practices and mood states mediated the effects of morningness-eveningness and menstrual distress on sleep quality. Our findings provide support for developing interventions to enhance sleep hygiene and maintain positive mood states to reduce the influence of neurophysiological factors on sleep quality among shift workers. Citation: Chung M-H, Liu W-I, Lee H-L, Hsu N (2013) Selected Neurophysiological, Psychological, and Behavioral Influences on Subjective Sleep Quality in Nurses: A Structure Equation Model. PLoS ONE 8(11): e79529. doi:10.1371/journal.pone.0079529 Editor: Allan Siegel, University of Medicine & Dentistry of NJ - New Jersey Medical School, United States of America Received July 28, 2013; Accepted September 28, 2013; Published November 20, 2013 Copyright: ß 2013 Chung et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors thank Dr. Chien-Hua Wu for his excellent statistical analysis supported by a grant from the Department of Health, Taiwan (DOH100-TD-B111-003). This study was supported by a grant (NSC99-2314-B-038-012-MY3) from the National Science Council, Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: minhuey300@tmu.edu.tw sleep quality, and (H2) a positive association exists between sleep hygiene practices and mood states. Psychological factors include reactions to stressful and emotional situations, such as anxiety and mood states, which are significantly associated with excessive worrying or depression during shift work [9]. Psychological symptoms related to neuroticism and depression were found to significantly increase from the late follicular to the late luteal phase in healthy females [10]. Mood states may be affected by menstrual distress. In addition, a previous study showed that poor sleep quality was significantly associated with negative mood states in a large cohort of college students [11]. Thus, we also hypothesized the following: (H3) a positive association exists between mood states and sleep quality, and (H4) a positive association exists between mood states and menstrual distress. Neurophysiological factors can be considered reactions to circadian variations and menstrual discomfort that are influenced by biological rhythms of sleeping and waking [12,13]. Morningness and eveningness refer to variations in the circadian phases of a person’s endogenous ‘‘clock’’ [14]. These diurnal types can be measured using the Morningness-Eveningness Questionnaire (MEQ), which distinguishes between morning (M)-type and evening (E)-type persons based on preferential behavioral rhythms in their daily activities and habitual sleep patterns [15]. According Introduction A considerable amount of evidence indicates that the effectiveness of non-drug treatments for insomnia is comparable to that of pharmacologic treatments [1,2]. Conceptual models for these nondrug treatments comprise behavioral, psychological, and neurophysiological constructs. Behavioral factors related to sleep hygiene may interfere with sleep quality [3]. Sleep hygiene is defined as the establishment of appropriate sleep behaviors that promote sleep quality [4]. These practices include maintaining a stable sleep schedule, not using the bed or bedroom for activities other than sleep, avoiding late-afternoon naps, and avoiding emotionally, physiologically, or cognitively stimulating activities before bedtime [4]. Insomnia patients frequently practice poor sleep hygiene such as smoking or drinking alcohol before bedtime [5]. Sleep hygiene practices may directly affect sleep quality. Moreover, certain people tend to experience numerous changes in their bedtimes, wake-up times, and durations of sleeping periods [6,7]. Maintaining lifestyle regularity was associated with a decreased risk of depression [8]. Adopting strategies, such as habitual sleep patterns, to improve sleep hygiene may reduce variations in mood. Thus, we hypothesized the following: (H1) a positive association exists between sleep hygiene practices and PLOS ONE | www.plosone.org 1 November 2013 | Volume 8 | Issue 11 | e79529 Sleep and Structure Equation Model Figure 1. Schematic representation of relationships among exogenous and endogenous variables (solid arrows represent hypothesized paths) that were examined using the structure equation model. doi:10.1371/journal.pone.0079529.g001 Taiwan. The inclusion criteria were as follows: (a) a registered female nurse who provided direct care for patients, (b) a full-time nurse who had worked rotating 8-h shifts for at least 3 months, and (c) no history of psychiatric or neurological disorders. Nurses who were pregnant at any time during the study were excluded. Of the 435 surveys distributed, 359 (82%) were returned, among which 21 were excluded for pregnancy and 2 were censored because of missing data. Thus, 338 completed questionnaires were analyzed. The questionnaire data assessed 19 parameters. Estimations of the response rate and a valid sample size indicated that at least 10,15 participants were required per measured indicator [19]. Our crosssectional analysis of the 338 nurses had a statistical power of 0.8. to the morningness-eveningness conceptual framework, people display different sleep hygiene practices. E-types experience numerous changes in their bedtimes, wake-up times, and durations of sleeping periods [6,7]. Chung et al. showed that morningnesseveningness is a significant predictor of sleep quality, and suggested that identifying a nurse’s diurnal type could assist in correcting poor sleep quality [16]. Furthermore, the morningnesseveningness preference of shift workers significantly influences their mood states [17]. Thus, we proposed the following hypotheses: (H5) a negative association exists between morningness-eveningness and sleep hygiene practices; (H6) a negative association exists between morningness-eveningness and sleep quality; and (H7) a negative association exists between morningness-eveningness and mood states. Women with severe menstrual symptoms are significantly more likely to use tobacco, drink alcohol heavily, and be overweight than women who do not have menstrual problems [18]. Smoking and drinking heavily close to bedtime are poor sleep hygiene practices [5]. Health behaviors related to sleep hygiene may be related to menstrual distress. In addition, women experiencing menstrual problems reported significantly increased distress, such as insomnia and fatigue [18]. Therefore, we proposed two additional hypotheses: (H8) a positive association exists between menstrual distress and sleep hygiene practice, and (H9) a positive association exists between menstrual distress and sleep quality. To date, most studies of the contributions of neurophysiological, behavioral, and psychological factors to sleep quality in nurses examined only isolated factors. Consequently, relationships among these factors in nurses remain unclear. In the present study, we used a structure equation model to examine contextual factors underlying sleep quality in nurses. We examined associations among behavioral factors of sleep hygiene, psychological factors of mood states, and neurophysiological factors involved in morningness-eveningness and menstrual distress and their effects on sleep quality. Ethical Considerations Our study was approved by the Institutional Review Board at Taipei Medical University Hospital. The purpose, content, duration, and methods of the study, as well as the guidelines for protecting their fundamental rights, were explained to potential participants before the commencement of the study during a ward meeting. All participants provided written consent. Data were anonymously collected, remained confidential, and were used for academic research purposes only. If a participant chose to withdraw during the course of the study, the researcher fully respected her right to do so, and guaranteed that her interests would not be harmed as a result. Instruments Morningness-Eveningness Questionnaire (MEQ). The MEQ is the most widely used questionnaire in chronopsychological research. It measures self-reported sleeping and waking habits [20]. People who display a preference for waking up early in the day are referred to as M-types. Conversely, E-types prefer sleeping later, and experience better functioning in the evening [21]. The MEQ contains 19 items, with possible total scores ranging 16,86. Our participants’ diurnal preferences were determined based on their MEQ scores. People who scored $59 points were categorized as M-types, and those scoring #41 points were categorized as E-types. Respondents scoring 42,58 points were considered neither type [20]. An acceptable internal consistency of 0.71 was reported for the Chinese version Materials and Methods Participants We performed a cross-sectional study using a convenience sample of nurses recruited from an urban regional hospital in PLOS ONE | www.plosone.org 2 November 2013 | Volume 8 | Issue 11 | e79529 Sleep and Structure Equation Model Table 1. Demographic characteristics, circadian types, menstrual distress, sleep hygiene, mood states, and sleep quality (N = 338). Sleep quality (mean ± SD) p value $26 8.0963.04 0.206 ,26 7.6363.45 Variable Table 2. Correlations among selected neurophysiological, psychological, and behavioral factors, and sleep quality (N = 338). BF Age (years) Nursing experience (years) $1.79 ,1.79 8.0563.02 0.220 7.6163.52 Body-mass index (kg/m2) $24 7.9263.28 ,24 7.3163.32 7.9263.30 Married 7.4263.23 7.8663.36 No 7.7163.01 7.8063.26 No 7.8763.34 7.3963.21 Evening shift 7.8263.62 Night shift 8.6962.94* 6.5362.63 Neither 7.8363.24 Evening 8.3063.57 0.865 ,0.05 0.064 Menstrual distress score $17 8.8063.39 ,17 6.8062.83 ,0.001 Sleep Hygiene Practice score $31 9.1263.19 ,31 6.4162.77 ,0.001 Profile of Mood States score $46 8.9763.20 ,46 6.4062.87 ,0.001 The dichotomized group was based on the median of each independent variable except for the body-mass index; SD, standard deviation of the mean; *p,0.05 vs day shift. doi:10.1371/journal.pone.0079529.t001 of the MEQ [22]. Cronbach’s a for our study was acceptable at 0.74, with a split-half reliability of 0.70. Short-Form Menstrual Distress Questionnaire (MDQ; MDQ-SF). The MDQ assesses a woman’s feelings during her menstrual cycle [23]. The questionnaire assesses 8 symptoms covered by 47 items. The MDQ demonstrated acceptable reliability and validity in previous studies, and the average a coefficient for each subscale [24] ranged 0.64,0.88. The MDQSF used in our study contains 22 items that assess 4 symptoms, including pain, water retention, autonomic reaction, and negative effects, on a 5-point scale [25]. A higher score indicates greater PLOS ONE | www.plosone.org 0.32* NF1 (morningness-eveningness score) 20.29** 20.15** 1.00 NF2 (menstrual distress score) 0.28* 0.41* 20.13* 1.00 SQ (sleep quality score) 0.49** 0.43* 20.13* 0.36* 1.00 1.00 insomnia patients with those of good sleepers [26]. The sleep hygiene practice section contains 19 self-reported items that inquire about how many days per week a participant practices certain activities, including napping, regular use of sleep medications, waking up at the same time every day, and smoking or drinking alcohol in the evening. Responses are scored on a 7-point Likert scale ranging from 0 (very beneficial) to 7 (very disruptive). Possible total scores range 0,133, and a higher score indicates poorer sleep hygiene practices [26]. Acceptable test-retest reliability was reported for the SHAPS [27]. In our study, the Chinese version of the SHAPS had a Cronbach’s a of 0.71 and a split-half reliability of 0.74 after the ‘‘sleep disturbance through temperature’’ item was discarded. Short-Form Profile of Mood States (POM-SF). We used the POMS-SF to assess mood states of participants during the week prior to data collection [28]. The 30-item questionnaire consists of tension, depression, anger, fatigue, confusion, and vigor subscales. Each subscale contains 5 items, each of which is scored on a 5-point Likert scale ranging from 0 (not at all) to 4 (extremely). Possible global scores range 0,120, and a higher score represents a higher level of mood disturbance [28,29]. The internal consistency of the English version of the POMS-SF [28] was shown to be at least 0.76. The Chinese version of the POMSSF respectively had internal consistency indices of 0.75,0.99 and 0.71,0.91 for patients with and those without pain. The content validity index was 0.92 [30]. Cronbach’s a for the 6 POMS-SF subscales in our study ranged 0.88,0.90. Pittsburgh Sleep Quality Index (PSQI). The PSQI measures self-reported sleep quality and sleep disturbances during the preceding month [31]. Its 7 components assess subjective sleep quality, sleep latency, sleep duration, sleep efficiency, sleep disturbances, use of sleeping medications, and daytime dysfunction. Each component is scored from 0 to 3, yielding a global score ranging 0,21, with a higher score indicating poorer sleep quality [31]. The reliability and validity of the PSQI were demonstrated in primary insomnia patients of various populations [32]. The PSQI has good internal consistency, with Cronbach’s a ranging 0.77,0.83 [33]. In studies of shift workers, Cronbach’s a for the PSQI was 0.70, indicating acceptable reliability [34]. The Chinese version of the PSQI demonstrated an internal consistency of 0.76 Circadian type Morning 1.00 PF (Mood States score) Sleep Hygiene Awareness and Practice Scale (SHAPS). The SHAPS compares sleep hygiene practices among 0.726 Shift schedule Day shift BF (Sleep Hygiene Practice score) SQ discomfort during menstruation. In our study, the MDQ-SF had a Cronbach’s a of 0.92 and a split-half reliability of 0.93. Consumption of coffee Yes NF2 0.284 Consumption of tea Yes NF1 BF, behavioral factors (sleep hygiene practice); PF, psychological factors (mood states); NF1, neurophysiological factor I (morningness-eveningness); NF2, neurophysiological factor II (menstrual distress); SQ, sleep quality. *p,0.05; **p,0.001. doi:10.1371/journal.pone.0079529.t002 0.238 Marital status Single PF 3 November 2013 | Volume 8 | Issue 11 | e79529 Sleep and Structure Equation Model Figure 2. Model of selected behavioral, psychological, and neurophysiological factors that affect sleep quality. Model fit indices: x2/ d.f. = 2.335, goodness of fit index (GFI) = 0.981, adjusted GFI (AGFI) = 0.947, comparative fit index (CFI) = 0.991, normed-fit index (NFI) = 0.985, root mean squared error of the approximation (RMSEA) = 0.063. Abbreviations: SHP, Sleep Hygiene Practices; POMS, Profile of Mood States; ME, morningness-eveningness; MD, menstrual distress; PSQI, Pittsburgh Sleep Quality Index; PSQI_D, PSQI in the day shift; PSQI_E, PSQI in the evening shift; PSQI_N, PSQI in night shift; error (e). * p,0.05. ** p,0.001. doi:10.1371/journal.pone.0079529.g002 [35]. In our study, the global PSQI had a Cronbach’s a of 0.71, indicating acceptable reliability. to determine significant predictors of sleep quality. The technique of parceling with an average of continuous multi-item scales was used to construct item parcels. For model fit and parameter estimations, a maximum-likelihood (ML) estimation was used, and measurement errors were included in the model. Measurement errors were computed for each latent variable as (12a)6d2, where a is the reliability estimate (Cronbach’s a coefficient) of the variable, and d2 is the variance of the mean score of the latent construct as previously described [36]. Parceled data are a few parameters to be estimated in defining a construct model and are considered to less likely have correlated residuals or multiple crossloadings [37]. Therefore, parceled data may result in reductions in various sources of sampling error. To determine the ML estimation of the model, values for the goodness-of-fit index (GFI), the adjusted GFI (AGFI), the normedfit index (NFI), the comparative fit index (CFI), and the root mean squared error of the approximation (RMSEA) were calculated. Statistical Analysis All analyses were performed using the SPSS computer software, vers. 20.0 for Windows (SPSS, Chicago, IL). Results of comparisons with a p value of ,0.05 were considered to represent statistically significant differences. Scores for the SHAPS, POMSSF, MEQ, MDQ, and PSQI were compared using Student’s t-test, and their relationships were explored using a Pearson correlation analysis. Fitness indices of the SPSS AMOS 20.0 (IBM, Armonk, NY) were used to estimate how well the hypothesized model fit the actual data. A model that adequately fits the data can provide a plausible representation of the causal structure of the study. Path diagrams were used to graphically represent the underlying structure of the variable effects in the hypothetical model (Figure 1). We used latent variable modeling with item parceling Table 3. Direct, indirect, and total effects of dominants on sleep quality. Variable Total effect Direct effect Indirect effect BF PF SQ BF PF SQ BF PF SQ NF1 (morningness-eveningness) 0.217 0.062 0.083 0.217 0.028 20.001 - 0.034 0.084 NF2 (menstrual distress) 0.345 0.441 0.364 0.345 0.386 0.139 - 0.055 0.225 BF (Sleep Hygiene Practice) - 0.158 0.353 - 0.158 0.311 - - 0.042 PF (Mood States) - - 0.267 - - 0.267 - - - BF, behavioral factors (sleep hygiene practice); PF, psychological factors (mood states); NF1, neurophysiological factor I (morningness-eveningness); NF2, neurophysiological factor II (menstrual distress); SQ, sleep quality. doi:10.1371/journal.pone.0079529.t003 PLOS ONE | www.plosone.org 4 November 2013 | Volume 8 | Issue 11 | e79529 Sleep and Structure Equation Model Table 4. Results of the mediation effect analysis. Equation Relationship Regression weight Standard error Sobel test 95% Asymmetrical coefficient interval NF1RBFRSQ NF1RBF 0.224 0.078 2.40* (0.06, 0.44) BFRSQ 1.005 0.230 NF2RBF 0.345 0.068 3.31* (0.15, 0.62) BFRSQ 1.005 0.230 NF2RPF 0.386 0.059 3.79* (0.15, 0.55) PFRSQ 0.861 0.185 BFRPF 0.158 0.072 1.98* (0.01, 0.31) PFRSQ 0.861 0.185 NF2RBFRSQ NF2RPFRSQ BFRPFRSQ BF, behavioral factors (sleep hygiene practice); PF, psychological factors (mood states); NF1, neurophysiological factor I (morningness-eveningness); NF2, neurophysiological factor II (menstrual distress); SQ, sleep quality. *p,0.05. doi:10.1371/journal.pone.0079529.t004 The GFI, AGFI, NFI, and CFI should achieve values of $0.90, and the RMSEA should be at least 0.08 if the model fits the data well [38]. We tested the hypothesis that psychological and behavioral factors predicted sleep quality through interactions with neurophysiological factors using a previously described method [39]. The Sobel test was used to estimate the significance of mediation effects [40]. In addition, the mediation effect on the product-of-coefficients model was assessed with asymmetric confidence intervals using the PRODCLIN2 program [41]. The 95% confidence intervals (CIs) did not include 0 to indicate the existence of mediation effects. In Figure 2, all path coefficients were significant and supported all of the hypotheses, except for H6 and H7. The 4 equations of indirect effects were used to examine mediation effects. As shown in Table 4, the NF1RBFRSQ, NF2RBFRSQ, and NF2RPFRSQ equations were used to estimate indirect effects of NF on sleep quality. Mediating effects exerted through these paths and the BFRPFRSQ equation were significant (Sobel test, p,0.05), and the 95% CI did not include 0, indicating mediation effects of behavioral and psychological factors on sleep quality. Results We tested a comprehensive conceptual framework for predicting the sleep quality of nurses. Statistical results supported the hypothesis that neurophysiological factors influence sleep by mediating psychological or behavioral regulation of normal sleep. Because there is limited empirical data from previous studies supporting the applicability of such a conceptual model, our results provide evidence of the applicability of the conceptual model among nurses. This is the first study to use a conceptual model to investigate behavioral, psychological, and neurophysiological factors underlying sleep quality in nurses. Our findings warrant further study of the conceptual model in other types of healthcare providers, in different clinical settings, and in different cultures. Our findings demonstrated that behavioral, psychological, and neurophysiological factors are significant predictors of sleep quality. In Table 3, our results showed that behavioral factors, such as sleep hygiene practices, caused direct and indirect effects on sleep quality. Moreover, behavioral factors appeared to be the strongest predictors of sleep quality (b = 0.31, p,0.001) as shown in Figure 2. Behavioral factors may be important predictors of sleep quality in nurses. Although it contrasts with those of some previous studies [12,42], this finding is consistent with those of others [43,44]. Sleep hygiene indicates that one maintains a regular sleep/wake pattern and controls all environmental factors that may interfere with sleep [4]. Nurses may know that shift patterns induce further irregular sleep-wake times; they usually try to practice good sleep hygiene. If they have difficulty sleeping at night because of work, they might limit themselves to more hours in bed at other times to reduce sleepiness on the night shift. The misalignment of biological rhythms with social rhythms imposed by one’s work schedule may explain why neurophysiological factors influence sleep quality through mediation of the regulation of sleep hygiene practice. Discussion Our study included 338 female shift nurses aged 27.8565.15 years, with an average of 3.5264.29 years of professional nursing experience. The average body-mass index was 20.9063.20 kg/ m2. Most participants were unmarried (82.0%). The majority of participants regularly drank tea or coffee (78.1% and 61.5%, respectively). Most of the participants were categorized as neither circadian type (71.9%). In Table 1, significant differences were found in sleep quality between groups of shift schedule, menstrual distress, sleep hygiene practice, and mood states. Therefore, the PSQI score in the structure equation model was measured by 3 shift groups. Table 2 shows that significant positive correlations existed for scores of sleep quality with mood states, sleep hygiene, and menstrual distress. However, a significant negative correlation was observed for scores of sleep quality with morningnesseveningness. As shown in Figure 2, the statistical goodness of fit of the structural model, denoted by the x2 value, was 2.335 (d.f. = 10, p = 0.010). The GFI was 0.981, the AGFI was 0.947, the NFI was 0.985, the CFI was 0.991, and the RMSEA was 0.063. In the model, sleep hygiene practice (b = 0.31, p,0.001), menstrual distress (b = 0.14, p,0.05), and mood states (b = 0.27, p,0.001) significantly predicted sleep quality. However, sleep quality was not significantly affected by the variable of morningness-eveningness (b = 0.00, p = 0.919). Table 3 summarizes direct, indirect, and total effects of dominant factors of sleep quality. According to path coefficients, sleep hygiene practices exhibited the strongest direct effects on sleep quality. Menstrual distress, despite showing a weaker direct effect than sleep hygiene, exhibited the strongest total effect on sleep quality. PLOS ONE | www.plosone.org 5 November 2013 | Volume 8 | Issue 11 | e79529 Sleep and Structure Equation Model Table 2 shows that mood states were positively correlated with menstrual distress and the PSQI score, such that poorer mood states were associated with higher menstrual distress and poorer sleep quality. Mood states also exerted direct influences on sleep quality. These results are consistent with those of a previous study, which showed that negative moods, such as depression and anxiety, were associated with nighttime sleep [45]. Furthermore, mood states mediated the effects of sleep hygiene practices on sleep quality as shown in Figure 2 and Table 4. Mood states may be affected by environmental and behavioral factors that are considered part of sleep hygiene practices, such as lighting, noise, temperature, hunger, caffeine intake, and the use of alcohol and tobacco. However, determining whether related factors of mood states are associated with sleep hygiene in predicting sleep quality requires further study. Menstrual distress, sleep hygiene, and mood states exerted direct effects on sleep quality as shown in Table 3. The regulation of sleep quality may be more affected by menstrual distress (total effect = 0.372) than by morningness-eveningness (total effect = 0.067; Table 3). Nurses likely experience poor sleep quality through indirect effects of neurophysiological factors, especially menstrual distress (b = 0.35, p,0.001; Figure 2). These findings are consistent with those of previous studies in which higher scores for menstrual distress correlated with higher PSQI scores, representing poorer sleep quality [46]. In addition, our results also showed that menstrual distress affected sleep quality by mediating mood states, and menstrual distress had stronger total effects on sleep quality. Menstrual distress indicates the intensity of symptoms that female nurses experience. Our results support a previous study [47] which showed that women with menstrual distress were significantly more likely to report frequent depression and insomnia than those without menstrual-related problems. These results imply that the effects of interventions on menstrual distress are important factors among female shift workers. The morningness-eveningness characteristics of nurses were associated with their mood states as shown in Table 2. This result is consistent with mood changes in morningness-eveningness chronotypes in healthy individuals [48]. However, our results showed that mediation effects of mood states were not significant between morningness-eveningness and sleep quality. One possible reason is that the neurophysiological factor of menstrual distress and behavioral factors of sleep hygiene were added to the structure equation model to decrease mediation effects. In Table 2, negative correlations were also found between mornings-eveningness and sleep quality. In regard to morningness-eveningness, participants who belonged to the E-type had worse sleep quality. This result is consistent with a previous study [49], which indicated that eveningness was associated with a shorter time in bed during the week. However, our results showed that morningness-eveningness had diminished effects on sleep quality through the mediator of sleep hygiene practice (b = 0.001, p = 0.919) in Figure 2. The stronger effects of sleep hygiene practice on sleep quality (b = 0.31, p,0.001) may explain why our findings were not consistent with those of a study by Chung et al. [16]. Clarification of the causal relationship between diurnal type and menstrual distress warrants further investigation. Certain limitations to our findings should be considered. Any causality suggested by the path analysis must be interpreted cautiously because of the cross-sectional design of our study. The POMS assesses mood states during the preceding week, whereas the PSQI assesses sleep quality during the preceding month. Thus, this difference in time frames may have caused some bias. The subjective scale of the SHAPS was not designed for shift workers, and does not address behaviors for promoting sleep that may be unique to shift workers, such as limited exposure to light during the night shift. In addition, not all neurophysiological and psychological factors were evaluated in our study. In future studies, researchers should consider differences in bedtimes resulting from habits or behaviors specifically related to the participants’ working conditions. Night shift workers may drink relatively high amounts of caffeine to assist them in staying awake during the night. Therefore, the amount and timing of caffeine ingestion should be recorded in future studies of shift workers. Our sample consisted of young nurses with relatively little work experience, all drawn from a single medical facility. These features of our study group limit the generalization of the results regarding the effects of diurnal preference, menstrual distress, mood states, and sleep hygiene on sleep quality in nurses working rotating shifts. Conclusions We examined possible pathways of associations among behavioral, psychological, and neurophysiological factors that mediate sleep quality in nurses. We evaluated the fitness of a conceptual model to clarify factors that influence sleep quality in nurses. Our results support the hypothesis that neurophysiological factors, such as menstrual distress and morningness-eveningness, influence sleep quality in nurses by mediating behavioral (sleep hygiene) and psychological factors (mood states). Future longitudinal studies are warranted to explore possible causal links between these factors. The clinical significance of our findings is that the effects of menstrual distress and morningness-eveningness on sleep quality may be reduced by practicing good sleep hygiene and maintaining positive mood states. Author Contributions Conceived and designed the experiments: HLL MHC. Performed the experiments: HLL MHC. Analyzed the data: WIL MHC. Contributed reagents/materials/analysis tools: NH MHC. Wrote the paper: MHC. References 6. Ishihara K, Miyake S, Miyasita A, Miyata Y (1988) Comparisons of sleep-wake habits of morning and evening types in Japanese worker sample. J Hum Ergol 17: 111–118. 7. Monk TH, Petrie SR, Hayes AJ, Kupfer DJ (1994) Regularity of daily life in relation to personality, age, gender, sleep quality and circadian rhythms. J Sleep Res 3: 196–205. 8. Furihata R, Uchiyama M, Takahashi S, Konno C, Suzuki M, et al. 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Two-period duopolies with forward markets Caleb Cox ∗ & Arzé Karam † & Matthias Pelster ‡. Abstract We experimentally consider a dynamic multi-period Cournot duopoly with a simultaneous option to manage financial risk and a real option to delay supply. The first option allows players to manage risk before uncertainty is realized, while the second allows managing risk after realization. In our setting, firms face a strategic dilemma: They must weigh the advantages of dealing with risk exposure against the disadvantages of higher competition. In theory, firms make strategic use of the hedging component, enhancing competition. Our experimental results support this theory, suggesting that hedging increases competition and negates duopoly profits even in a simultaneous setting. Keywords: Corporate hedging, duopoly, dynamic setting, game theory, laboratory experiments, strategic application. JEL Classification: D21, D22, D43, D53. ∗ Virginia Commonwealth University, Snead Hall, 301 W. Main Street, Box 844000, Richmond, VA, USA 23284. Phone: +1 804 828 7101. Fax: +1 804 828 9103 E-mail:cacox@vcu.edu. † Durham University, Mill Hill Lane, DH13LB, England. Phone: +44 191 33 45460. Fax: +44 191 3345201. E-mail:arze.karam@durham.ac.uk. ‡ Paderborn University, Center for Risk Management. Warburger Str. 100, 33098 Paderborn, Germany. Phone: +49 5251 60-3766. Fax: +49 5251 60-4207. E-mail: matthias.pelster@upb.de 1 Introduction Corporate hedging is of great interest to both practitioners and academics (e.g., Graham and Rogers, 2002; Haushalter, 2000; Mello and Parsons, 2000; Tufano, 1996; Smith and Stulz, 1985). Managing risks can reduce firms’ expected bankruptcy costs, agency costs, information asymmetries, or expected taxes, and thus increase shareholder value (e.g., Bolton et al., 2011; Campello et al., 2011; Froot et al., 1993; Mackay and Moeller, 2007).1 While several optimal strategies developed theoretically propose a full hedge by firms in forward markets (e.g., Broll and Wong, 2013; Holthausen, 1979), several empirical studies find underhedging: hedge ratios that are less than one (e.g., Adam et al., 2015; Brown et al., 2006; Carter et al., 2006; Haushalter, 2000; Tufano, 1996). A “sequential” setting where firms first decide on their hedging and then set their production quantity provides a theoretical rationale for underhedging in imperfectly competitive markets (Broll et al., 2009). In such cases, firms account for the effect that their hedging decision may have on the market (Brandts et al., 2008; van Eijkel and Moraga-Gonzalez, 2010; Leautier and Rochet, 2014; Le Coq and Orzen, 2006). Ceteris paribus, a larger hedging position of one firm increases the optimal output of this firm and decreases the competitors’ optimal output (Allaz, 1992; Allaz and Vila, 1993; Broll et al., 2011). In equilibrium, however, this increases competition: Output increases, and prices decrease. Thus, firms may decide to hedge less to avoid reducing their profits. It is clear that firms face a strategic dilemma: They must weigh the benefits of hedging their risk against the adverse effects of an increase in market competition. However, Broll et al. (2011) theoretically show that this strategic dilemma is non-existent in a simultaneous one-shot output market interaction setting where firms decide on their production and hedging decision at the same time. In this paper, our main objective is to examine experimentally the extent to which strategic considerations explain underhedging behavior in a simultaneous hedging setting in a multi-period framework in imperfectly competitive markets. We use a simple Cournot duopoly model with a simultaneous hedging setting and repeated interaction to examine how hedging affects market equilibrium. Ex ante, we first allow firms to hedge their risk exposure toward demand uncertainty on a forward market. Second, we provide firms with the real option for storage that allows them to react to 1 For literature discussing the goal of firms employing corporate hedging see, among others, Géczy et al. (1997); Kajüter (2012). 1 low demand realizations ex post. This latter feature introduces real dynamics into the multi-period model and ensures that our setting is not simply a repetition of the single-shot game. In our experimental study, we conduct two treatments: one treatment with an additional production opportunity after the initial demand state is realized (Double Production), and one treatment without (Single Production). Subjects play repeatedly for 20 rounds to allow them to learn about the strategic setting, but they are randomly re-matched in each round to minimize any potential repeated game effects. Our experimental results provide supportive evidence that subjects tend to account for the adverse effects of their own financial decisions on the market equilibrium. However, we do not find any evidence that the experimental decision makers consider the hedging decisions of their competitors – which are common knowledge – in subsequent decisions. As the game is closer to a simple repetition of the single-shot game in our second setting, subjects’ level of supply prevents duopoly profits, on average. Our results underline that hedging creates a strategic dilemma for producing firms and significantly increases competition—even in a simultaneous setting. 2 Risk-sharing markets and competitiveness in duopoly markets According to the principle of increasing uncertainty, the risk-averse firm will produce less under (demand) uncertainty than it would under certainty (Leland, 1972; Sandmo, 1971). However, with an access to risk-sharing markets such as forward markets, the decision to produce is not subject to risk considerations. The reason is that forward markets allow the firm to buy or sell contracts for future delivery and thus, hedge and manage its risk exposure. As a result, the production decision of the competitive firm is separated from the forward trading decision and should match the production amount under certainty (Feder et al., 1980). This result indicates that risk-sharing markets provide an important social benefit by offering the opportunity to reduce or even eliminate output fluctuations that are due to the variation in firms’ subjective distributions of uncertainty. While risk-sharing markets increase the production and profits for a competitive firm, this does not necessarily apply to a duopolistic (oligopolistic) market. Duopolistic markets are characterized by an inverse relationship between the cumulative production 2 of the competing firms and the market price of the offered product. As a result, firms may generate higher revenues and profits by reducing their production in response to an increasing market price. The intuition here is that lower production eases competition in the market and allows firms to realize higher prices. Similar to the competitive firm under uncertainty, risk-averse firms in a duopoly also decrease their production in Nash Equilibrium (NE) when facing demand uncertainty. Lower production yields higher prices, and consequently firms may increase their revenues and profits under uncertainty, compared to the certainty case (Eldor and Zilcha, 1990). In such a setting, forward markets may increase the competition between duopolistic firms via two channels. First, forward markets allow the firms to manage the risk exposure of their production decisions. Being able to manage their risk exposure not only by reducing their production, but also via the risk-sharing markets, firms may as a result increase their production. Second, a firm can credibly establish its intention to provide a large supply, which thereby (ceteris paribus) increases its own optimal output and decreases the competitors’ optimal output (Broll et al., 2011). As noted by Wolak and McRae (2009), the larger is a supplier’s fixed forward contract obligation, the larger is its best-reply output level. Forward sales reduce the residual demand that face the firm, and thus reduce the gain from restricting output to increase price (Wolak and McRae, 2009). Consequently, forward sales reduce the incentive to withhold output. Both channels yield an increase in NE output. However, the firms are not necessarily better off. The introduction of risk-sharing markets have two opposing effects. First, the risk-averse firm improves its position by reducing riskiness via risk-sharing markets. Second, however, the increased production results in lower market prices. Eldor and Zilcha (1990) show that although firms are risk-averse, in some cases risk-sharing markets make all firms worse off in the NE. Firms must thus weigh the reduced risk of forward sales against the accompanying reduction in profitability. 3 Related literature Following the seminal work of Allaz (1992) and Allaz and Vila (1993), several experimental studies investigate the strategic impact of a hedging device on market competition. Importantly, one has to distinguish two settings. First, in a simultaneous setting, hedging and output decisions are taken at 3 the same point in time. According to Broll et al. (2011), in this setting, hedging is exclusively used for risk-managing reasons as it is not possible to use hedging strategically. Second, in a sequential setting, the hedging decision is made before the output decisions. In this setting, hedging is not only used to manage the risk exposure but also as a strategic device and increases competition (Allaz and Vila, 1993; Broll et al., 2009; Wolak and McRae, 2009). The competition-enhancing effect of selling forward has been tested in experimental Cournot duopolies several times. Based on Allaz and Vila (1993), Le Coq and Orzen (2006) study a single forward and a spot market phase. First subjects sell their product in the forward period, before they compete on the residual demand in the subsequent spot period. These experimental results show that the introduction of forward markets does have competition-enhancing effects, which are, however, not as strong as theory predicts. Motivated by the specific design of forward markets that occur in the electric power industry, Brandts et al. (2008) consider both quantity and supply function competition as strategic variables. For both types of competition, they find that the introduction of a forward market significantly increases competition, as is predicted by the model of Allaz and Vila and in line with the notion of Wolak and McRae (2009). Closely related, van Koten and Ortmann (2013) show that forward markets are a more effective remedy to increase competition compared to increasing the number of competitors. Our study contributes to this literature by allowing players to utilize the risk management purpose of the hedging device; they thus face a dilemma between hedging their risk exposure and maintaining high market prices. The previous experimental studies discussed here exclusively focus on the strategic impact of the hedging device and abstract from risk management considerations, as their settings do not incorporate risk. Thus, our study is closer to the theoretical setting of Broll et al. (2011), which accounts for risk, than to the theoretical setting of Allaz and Vila (1993). Hence, our setting is cognitively more complex than the setting of Allaz and Vila, as decision makers have to account for the risk that is associated with their decisions. In addition to experimental studies, some contributions use field data and provide empirical evidence of the topic. Wolak (2000) shows that, for the Australian power market, the effect is pro-competitive when firms use the forward market (see also Wolak and McRae, 2009). Similarly, van Eijkel and 4 Moraga-Gonzalez (2010) study the Dutch wholesale market for natural gas and show that strategic reasons play an important role in explaining the observed firms’ hedge ratios. However, these studies are unable to separate the market views of managers from strategic applications of the hedging device. Our laboratory setting allows us to implement the desired variations with a high degree of control. 4 Hypotheses development We experimentally study the strategic considerations of corporate hedging in a simultaneous setting. For this, we use a simple Cournot duopoly model with repeated interaction. We restrict ourselves to the Cournot setting, as Kreps and Scheinkman (1983) argue that quantity precommitment and Bertrand competition yield Cournot outcomes. In other words, in an oligopoly under mild assumptions about demand, the unique equilibrium outcome is the Cournot outcome.2 We consider two different settings, Single Production and Double Production. 4.1 Single Production setting Our basic experimental setting considers a two-period model without discounting. The first period runs from time t = 0 to time t = 1; the second period stretches from t = 1 to t = 2. Two symmetric firms—firm A and firm B—decide to produce a homogeneous good, qtA ≥ 0 in t = 0. Production takes place between t = 0 and t = 1 and gives rise to unit costs of 1 in t = 1. Each firm’s production becomes common knowledge in t = 1. In t = 1 and t = 2 firms have the opportunity to sell their production on the same market. The demand for the homogeneous good is uncertain. The price in time t is determined by the inverse B demand function, p̃t = ε̃t − (sA t + st ), where ε̃t expresses the uncertainty of the realization of demand and sA t denotes the supply of firm A in time t. The initial random demand state ε1 is drawn from {40, 60} with equal probability. The subsequent random demand state ε2 is drawn 2 Even though the Cournot model has been criticized for its theoretical foundations, the model is simple and has pleasing comparative statics and has proved useful in the literature (see, e.g. Mas-Colell et al., 1995; Martin, 1994; Maggi, 1996; Larue and Yapo, 2000). 5 demand for the homogeneous good is uncertain. The price at time t is determined by the inverse demand function, p̃t = ε̃t − (s1 + s2 ), where ε̃t expresses the uncertainty of the saturation of demand and si denotes the supply of firm i. The initial random demand state ε1 is drawn from {40, 60} with equal probability. The subsequent random demand state ε2 is drawn with equal probability with equal from {50, 70} conditional the high initial demand state,onorlow from {30, 50} from probability {50, 70} conditional on high initial demandonstate, or from {30, 50} conditional initial demand Figure 2 depicts the uncertainty of demand. conditional on state. the low initial demand state. Figure 1 depicts the uncertainty of demand. 70 60 today 50 40 30 t=0 t=1 t=2 Figure 1: Evolution of random variables Figure 1: Evolution of random variables 5 As firms can sell their production in t = 1 and t = 2, they are endowed with the real option to distribute their production between periods. This allows firms to react to bad price realizations in t = 1 due to low demand and store their production (or parts of their production) to sell in t = 2. In t = 1, with knowledge of the actual realization of demand for this period, firms make their sales decision (s1 ) and, thus, also their storage decision (q1 − s1 ). Sales and inventory have to add up to the production amount. In addition, firms are able to ex ante manage their exposure to price risk via an unbiased forward market.3 The forward market offers contracts with a maturity of one period.4 Hence, firms can purchase or sell forward contracts on the good that they produce in time t = 0 with maturity in t = 1 and in t = 1 with maturity in t = 2. Binding first-period hedging decisions (h1 ) are made simultaneously with the production decision in t = 0; second-period hedging decisions (h2 ) are made in t = 1 and manifest in t = 2. As the natural risk management decision for a producing firm is to sell its product on the forward market, h > 0 denotes a short position in our context. As firms that take a long position on the forward market are increasing their risk exposure, we do not allow h < 0.5 3 The forward market is called unbiased if the forward price equals to the expected spot price. Since in reality forward contracts also have limited maturities, we do not include contracts with a maturity of two periods or longer in our model. 5 Note that this is only relevant if decision makers are risk-neutral or even risk-seeking. 4 6 Similar to the spot price, the forward price is the result of expected demand and expected (equilibrium) decisions of the duopolists: the supply. Forward-market participants act rationally and have perfect knowledge.6 As a result, they are aware of the optimal decisions of the duopoly and act accordingly.7 Consequently, the one-period forward price (which is agreed on in t = 0, and is executed in t = 1) is given by B A B f (0, 1) = E[ε̃1 ] − (ŝA 1 + ŝ1 ) = 50 − (ŝ1 + ŝ1 ), (1) B where ŝ1 = sSP (q1A , q1B , hA 1 , h1 ) denotes the (anticipated) equilibrium choices of producing firms for a given subgame-path.8 The forward rate for second-period contracts is uncertain in time t = 0; only the probability distribution of the forward rate is known. As the distribution of the random demand state ε2 is conditional on the initial demand state, the second-period forward rate relates to the spot rate in t = 1. As a result, the forward price for the second period fulfills f˜(1, 2) = E1 [ε̃2 | ε1 ] − industry supply B = ε̃1 − (q̌1A + q̌1B − sA 1 − s1 ), where .̌ denotes realized choices from previous interactions. In t = 1, the demand uncertainty for the first period is resolved. Based on the demand state firms choose their supply for the spot market (s1 ), which is sold immediately. The remainder of the production is stored and will be sold in t = 2. Warehouse charges are 0.25 per unit, for every unit that is produced in the first period and sold in the second period. Additionally, firms decide on their second-period hedging (h2 ) in t = 1.9 6 Besides demanders for the homogeneous good, speculators may have incentives to engage in forward trading and take offsetting positions of the firms. 7 Cyert and DeGroot (1970) make a similar argument, that the counterpart’s choices cannot be observed in a simultaneous-move game and, hence, subjective expectations have to be considered. 8 We provide the first-period forward price that take the equilibrium choices into account in Equation (5) below. 9 Note, that at this point we do not allow firms to enter production in time t = 1. Hence, firms cannot produce additional goods for the second period. Possible justifications for this limitation include the possibility to produce large lot sizes, high setup costs, or seasonal limitations in production. 7 In t = 2, the demand uncertainty for the second period is resolved and the remaining production is sold. As all goods have to be sold at this point, second-period sales are an immediate result of the A A firm’s previous decisions (sA 2 = q1 − s1 ). Therefore, second-period sales do not represent a decision variable. Figure 2 visualizes the timing of the decisions. Demand ε1 Realisation of Random Variables Demand ε2 Collecting Profits Time Decision Variables 0 Production q1 Hedging h1 Profit π1 Profit π2 1 2 Hedging h2 Sales s1 Figure 2: Single Production setting The profits of the firm are the sum of period 1 and 2 profits, as given by profit period 1 = spot market price1 · supply1 − production costs + profits or losses from hedging decision1 − storage costs (2) and profit period 2 = spot market price2 · supply2 + profits or losses from hedging decision2 (3) where profits / losses from the hedging decision are given by profits or losses from hedging decision = hedging amount · (forward price − spot price). 8 Mathematically, profits of firm A (B) are given by π̃ A = π̃1A + π̃2A with B A A A A A π̃1A = (ε̃1 − (sA 1 + s1 ))s1 − q1 + h1 (f (0, 1) − p̃1 ) − 0.25(q1 − s1 ) and B A A A ˜ π̃2A = (ε̃2 − (q1A + q1B − (sA 1 + s1 )))(q1 − s1 ) + h2 (f (1, 2) − p̃2 ), A A B A B where sA 1 = s1 (q̌1 , q̌1 , ȟ1 , ȟ1 ). That is, the supply in t = 1 is a function of the decisions that were A made in the previous stage. Additionally, sA 1 ∈ [0, q̌1 ]. As the duopoly is symmetric, firm B has the same profit functions. In order to determine forward prices and derive hypotheses about participants expected choices, we assume that both firms maximize (µ, σ)-preferences. Here, µ = E[π̃ A ] denotes the expected value and σ 2 = var(π̃ A ) the variance of the stochastic profit of firm A. We rely on the (µ, σ)-approach as it provides a simple approach to complex problems and consequently a cost-efficient approach to information.10 Thus, the decision rule of firm A (B) is given by A ΦA = µA 1 + µ2 − αA ((σ1A )2 + (σ2A )2 ). 2 The preference value of the firm increases in the expected profits across both periods and decreases in the variance of those profits. Parameter αA denotes the degree of risk aversion of firm A and specifies the sensitivity of the firm reactions to the variance of its profits.11 Note that αA ≡ 0 denotes risk neutrality—and thus the situation under certainty. (Since participants received all payments at a single point in time (the end of the session), time preferences play no role in the experiment.) Thus, we abstract away from discounting. All parameters are common knowledge. We focus on subgame-perfect Nash equilibrium (Selten, 1965) in a Cournot setting. The strategy choice consists of the optimal production and hedging decisions as well as strategy sets for the supply that constitute a Cournot-Nash equilibrium for every possible production and forward decision. We first have to find the Cournot-Nash equilibrium in t = 1 for all possible production and forward decisions. Then, we go back to t = 0 and determine the optimal output and hedging decisions which consider the effects on the quantity sold in t = 1 as well as the subsequent hedging decision. A 10 11 For more detailed justification of the (µ, σ)-approach see, e.g., Robison and Barry (1987). The preference function describes risk-averse behavior as long as utility increases in expected profits and decreases in risk and marginal utility in expected profits does not increase (∂Φ/∂µ > 0, ∂Φ/∂σ < 0, and ∂ 2 Φ/∂µ2 ≤ 0). 9 detailed numerical example is provided in Appendix A. In a subgame-perfect Nash equilibrium, the optimal supply decision in t = 1 fulfills (given the realization of demand uncertainty ε1 ) B A A A B B ε1 − (2sA 1 + s1 ) + ȟ1 = ε1 − (2q̌1 − 2s1 + q̌1 − s1 ) − 0.25. (4) The first part of the left-hand side of the equation denotes the marginal revenues from the spot sales in t = 1, while the second part of the left-hand side (ȟA 1 ) denotes the marginal revenues from the hedging decision that was made in t = 0 and is executed in t = 1. The right-hand side of the equation denotes the marginal revenues from spot sales in t = 2, adjusted for the costs that result from the shift of sales: the costs for storage 0.25. Thus, the intuition of Equation (4) is that decision makers seek to smooth their marginal revenues over the periods. For the optimal decision, the marginal revenues that are realized in t = 1—the left-hand side of the equation equal the marginal revenues that are realized in t = 2: the right-hand side of the equation. Note, that the forward-market decision for the second period does not directly influence the sales decision in t = 1. However, the existence of a forward market enables firms to separate their sales decision in t = 1 from their risk preferences α, which do not appear in Equation (4). In Equation (4), ȟA 1 shows that the optimal sales decision is influenced by the hedging decision that is made in t = 0. This highlights the strategic impact of the hedging decision in a simultaneous setting. The reason is that hedging increases the quantities that are supplied and thus effectively increases the vigor of competition: The decision maker increases her supply on the spot market with A her own forward position (∂ŝA 1 /∂h1 = 1/3, see Equation (17) in Appendix A) and decreases her B supply on the spot market with her competitors forward position (∂ŝA 1 /∂h1 = −1/6, see Equation (17) in Appendix A). As the forward price is fixed and not subject to the quantity that is offered on the spot market, the decision maker is less sensitive towards price changes due to additional supply (see also Wolak and McRae, 2009). Hence, with a larger forward position the decision maker also increases supply in t = 1. With respect to the equilibrium solution, the first-period forward price for the hedging decision that 10 is made in t = 0 and executed in t = 1 reads 1 1 1 B f (0, 1) = 50 − (q1A + q1B ) − (hA · 0.25. 1 + h1 ) − 2 6 3 (5) The forward price reflects that the market supply in t = 1 depends on the firms’ production decisions and firms’ initial hedging decisions. Hence, the forward price decreases in the firms’ production decisions and in their hedging decisions. Finally, the forward price decreases in the costs that result from the shift of sales (costs for storage, 0.25) from t = 1 to t = 2, as the market supply in t = 1 increases in costs for the storage for goods that are not sold in t = 1. Next, we consider the hedging decision that is made in t = 1 and executed in t = 2. In equilibrium, the producers will sell the entire production that is taken into storage on the forward market (see Equation (16) in Appendix A. This result is consistent with the observation that any risk-averse decision maker—independent of the degree of risk aversion—will always prefer the safe choice over the lottery as long as both have the same expected value. Thus, the profit that a firm realizes in t = 2 becomes deterministic in t = 1. Given the equilibrium decisions in the second period, we turn to the equilibrium in t = 0. Here, we obtain several insights: First, decision makers cannot separate their production and their risk management decisions in t = 0. Their production decision depends on their risk preferences α and their expectations about the uncertainty. Therefore, production is reduced, as compared to the duopoly setting under certainty. Second, firms account for the strategic impact of the hedging decision. The hedging decision depends on risk preferences, on the expected uncertainty, and on the effect of the hedging choice on the future sales decisions of the firm and of its competitor. As was shown above, the equilibrium sales decisions of firms are subject to the hedging decision that is made in t = 0. The equilibrium industry supply increases with every additional forward contract that decision makers engage in, which yields lower market prices. Thus, decision makers have to balance the reduced risk exposure that is due to a larger hedging position (the risk management aspect) against the revenue potential of their sales. Decision makers that focus only on the risk management aspect will engage in large hedging positions while those who only focus on the potential revenue and not on the risk will take smaller hedging 11 positions. Due to the balancing of these extreme behaviors, we expect that decision makers will choose an underhedge in time t = 0 (which follows from Equations (18) and (19) in Appendix A). To summarize, we hypothesize that 1a. Firms will make their sales decision in an effort to equate marginal revenues over periods (see Equation (4)). They will attempt to equate the marginal revenues of the first period with the marginal revenues of the second period, adjusted for storage costs. 2a. Sales decisions are independent of degree of risk aversion. The existence of a forward market allows players to separate their sales decision in the first period from their risk preferences (see Equation (4)). 3a. Firms consider previous hedging choices when they make their sales decisions. Firms spot sales in t = 1 increase in their own first-period hedging choice with a marginal effect of +1/3, while spot sales in t = 1 decrease in the first-period hedging choice of the competitor with a marginal effect of −1/6 (see Equation (17) in Appendix A). 4a. In the second period, firms will sell the entire production that had been taken into storage on the forward market: Firms will take a full hedge. Their hedging decision is independent of their risk aversion. 5a. In the first period, firms will take a hedging position that increases in their risk aversion and is smaller than their expected sales: Firms will choose an underhedge. 4.2 Double Production setting Next, we turn to the Double Production setting, which is similar to the Single Production setting— except for one crucial difference: Firms can make an additional production decision in t = 1 for production that will be available for sale in the second period: between t = 1 and t = 2. Again, decision makers have the opportunity to react to low demand realizations by storing their good and selling at a later time. Due to the adjustment, the new decision sequence takes the following form: In t = 0, firms set their production for the first period as well as their forward position. Production takes place between 12 Demand ε1 Realisation of Random Variables Demand ε2 Collecting Profits Time Decision Variables 0 Production q1 Hedging h1 Profit π1 Profit π2 1 2 Production q2 Hedging h2 Sales s1 Figure 3: Double Production setting t = 0 and t = 1. In t = 1, demand uncertainty for the first period is resolved. Firms choose their sales for t = 1. Excess production is taken into storage. Also, the forward position from the first period is closed. Moreover in t = 1, firms set their production for the second period and their forward position with maturity in t = 2 (which is agreed on in t = 1, and is executed in t = 2). The demand in t = 2 is still uncertain at this point. Again, production takes place between t = 1 and t = 2. At the final date (t = 2), firms sell their additional production and everything from storage. Figure 3 visualizes the setup of the model. Consequently, the profit of firm A (B) in t = 2 is given by profit period 2 = spot market price2 · supply2 − production costs2 + profits or losses from hedging decision2 , (6) and, mathematically, by ˜ π̃2A = p̃2 (Q2 )(q1A − sA + q2A ) − q2A + hA 2 (f (1, 2) − p̃2 ). 13 (7) The first-period profit remains unchanged, A A π̃1A = p̃1 (Q1 )sA − q1A + hA 1 (f (0, 1) − p̃1 ) − 0.25(q1 − s ). (8) The inverse demand function in time t is still given by p̃t (Qt ) = ε̃t − Qt , where Qt denotes the B A B A B A B industry supply in time t. Specifically: Q1 = sA 1 + s1 and Q2 = q1 + q1 + q2 + q2 − (s1 + s1 ), respectively. As in the Single Production setting, the forward prices are determined by (expected) demand and (anticipated) supply. For the second period f˜(1, 2) = E1 [ε̃2 | ε1 ] − industry supply B A B = ε̃1 − (q̌1A + q̌1B − ŝA 1 − ŝ1 + q̂2 + q̂2 ). Next, we determine the subgame-perfect Nash equilibrium. For time t = 1 decisions, the firm chooses its second-period production by taking into account the amount that remains in stock so that the marginal costs equal the marginal revenues. Its decision can be separated from its degree of risk aversion (α does not enter Equation (9)): B B B E[ε̃2 ] − 2(q2A + q̌1A − sA 1 ) + (q2 + q̌1 − s ) = 1. (9) Moreover, the amount in stock immediately relates to the amount to be sold on the spot market in t = 1. The firm chooses the amount to be sold on the spot market in t = 1 to smooth marginal revenues over time: The realized marginal revenues from period one equal the marginal revenues in the second period, corrected for storage costs and adjusted with respect to the risk management decision taken in t = 0: B A A A A B B B ε1 − (2sA 1 + s1 ) + ȟ1 = ε1 − 2(q2 + q̌1 − s1 ) + (q2 + q̌1 − s1 ) − 0.25 (10) = 1 − 0.25 = 0.75. The first part of the left-hand side of the equation denotes the marginal revenues from the spot sales in t = 1, while the second part of the left-hand side (ȟA 1 ) denotes the marginal revenues from 14 the hedging decision that was made in t = 0 and is executed in t = 1. The right-hand side of the equation denotes marginal revenues from the sales in t = 2, adjusted for costs resulting from the shift of sales (costs for storage = 0.25). Due to the additional production opportunity and the hedging decision that is made in t = 1, the marginal revenues from sales in t = 2 equate to the marginal costs from production (see Equation (9)). As a result of the risk-sharing markets, the decision is independent of the degree of risk aversion of the firm, α. Additionally, Equation (10) indicates that the optimal sales decision is influenced by the hedging decision (ȟA 1 ) that is made in t = 0, which highlights the strategic effect of the hedging opportunity.12 In t = 1, the uncertain demand from the first period is realized, which determines the current spot price. Also, the forward price for the second-period hedging choice—which firms make now and will be manifested in t = 2—is known. Hence, the firm can set its supply for both periods in a certainty-like situation. The optimal forward position in t = 1 can be determined in accordance with the full-hedge theorem: The firm sells its entire stock and additional production forward in t = 1: A A A hA∗ 2 = q2 + q̌1 − s1 (11) As a result, profits in t = 2 are deterministic. The competition for both periods takes place at the same time and follows the basic rules known from Cournot duopoly: If the competitor increases its supply by one unit, the firm reacts by decreasing its own supply by 1/2. The equilibrium corresponds to the equilibrium from the one-shot Cournot duopoly under certainty. The ability to take the realized spot price into account crucially depends on the ability to store the good. Due to the storage option, the produced quantity and the supplied quantity may differ. Thus, the decision for the sales on the spot market can be made when the spot price is known. Clearly, this is not possible without the possibility to store the production good. The amount of additional production, B q2A (q̌1A , ȟA 1 , ȟ1 ) = 12 E[ε2 ] − 1 − (q̌1A − sA 1) 3 Note that Equation (10) is equivalent to Equation (4) from the Single Production setting in Section 4.1. 15 (12) reflects the quantity that is known from the single-shot game corrected for stored goods. We obtain the well known example from game theory: In a repeated game the result of the final interaction corresponds to the result of the single-shot game (see, e.g., Vives, 1999). Turning to the influence of the hedging decision on sales, we observe that hedging increases competition as it does in the Single Production setting: As firms increase their hedging position (= larger short position), the supply on the spot market increases due to the strategic impact of the hedging device. As a result, spot prices decrease. The main decisions in t = 1 remain unchanged, as the firm smooths its marginal revenues over time—depending on its time preferences and storage costs. However, the firm can now adjust marginal revenues for the second period to marginal costs for the second period due to the ability to adjust production. This ability increases competition, as the second-period interaction effectively occurs under certainty. Turning to the decision process in t = 0, we again observe that the firm cannot separate real and financial decisions. The firm chooses its initial production according to its intended sales. Intended sales are set in an effort to equate marginal revenue and marginal costs as the subsequent decision in t = 1 essentially takes place under certainty. Hence, firms do not use the option to take storage to be able to react to high demand realizations. However, ex post excess production can happen: If the realization of demand is not as expected but below the expected amount, firms react by putting current production into storage and decreasing their current supply. With respect to the initial hedging decision, the strategic impact of the hedging component on competition in t = 1 again comes into play. Firms compete via their risk management decision, and thus account for the strategic impact of their hedging decision. The optimal hedging choice equals expected spot sales in t = 1 corrected for the impact on competition. The magnitude of correction depends on risk aversion and price risk. Thus, in t = 0, decision makers will choose an underhedge. Hence, forward markets increase competition in oligopolistic markets in two ways: First, the decision maker is able to deal with uncertainty via forward markets which in itself increases the industry supply. The industry supply equals the industry supply under certainty. Second, the strategic impact and the fact that the first-period forward price is agreed on in t = 0 and does not react to 16 the increased supply, reduces the gain from restricting the spot-market output and thus increases industry supply even further (see also Wolak and McRae, 2009). Last, we compare the Double Production setting with the Single Production setting: The main difference between the settings is the firms’ access to risk-sharing markets to manage the risk exposure for their supply in t = 2. In the Single Production setting firms’ supply in t = 2 depends on their initial production decision, which they have to set in t = 0 without having access to risksharing markets for both periods. Firms can manage the risk exposure of sales made in t = 1; however, forward contracts with maturity in t = 2 become available only in t = 1. In the Double Production setting, however, firms can decide on their supply in t = 2 when setting their secondperiod production in t = 1, while having access to forward contracts to manage the risk exposure immediately. Hence, firms do not have to rely on initial production (from t = 0) to supply the market in t = 2. As a result, second-period supply will be larger in Double Production and the market will be more competitive. As firms equate marginal revenues in t = 1 with marginal revenues in t = 0 in both settings, the first-period spot market is more competitive in the Double Production setting as well, which is a result of the additional production decision’s being detached from demand uncertainty. To summarize, we hypothesize that 1b. Firms will make their sales decision in an effort to smooth marginal revenues over periods. They will aim for the marginal revenues of the first period to equal the marginal revenues of the second period, adjusted for storage costs. 2b. Sales decisions are independent of the degree of risk aversion. The existence of a forward market allows firms to separate their sales decision in the first period from their risk preferences. 3b. Firms consider previous hedging choices when making sales decisions. Firms’ spot sales in t = 1 increase in their own first-period hedging choice with a marginal effect of +2/3, while spot sales in t = 1 decrease in the first-period hedging choice of the competitor with a marginal effect of −1/3 (see Appendix A, Equation (23)). 17 4b. In the second period, firms will sell their entire production that has been taken into storage and the additional production on the forward market: Firms will take a full hedge. Their hedging decision is independent from their risk aversion. 5b. In the first period, firms will take a hedging position that depends on their risk aversion and is smaller than their expected sales: Firms will choose an underhedge. 6. Spot sales in t = 1 in the Double Production setting will be larger than in the Single Production setting. Table 1 summarizes the definitions of the decision variables and the other variables that are used in our study. Table 2 summarizes the treatments. Table 1: Variable definitions Variable Variable name Definition q1 quantity quantity produced in the first period, decision in t = 0. q2 quantity2 quantity produced in the second period, decision in t = 1. otherquantity quantity produced by the competitor in the first period, decision in t = 0. s1 spot sales1 quantity of sales in the first period, decision in t = 1. s2 spot sales2 quantity available for sale in the second period, immediately follows from decision in t = 1. h1 hedging1 quantity hedged in the first period, decision in t = 0. h2 hedging2 quantity hedged in the second period, decision in t = 1. otherhedging1 quantity hedged by the competitor in the first period, decision in t = 0. otherhedging2 quantity hedged by the competitor in the second period, decision in t = 1. highstate indicator equal to one if the high demand realization occurs in the first period, zero otherwise. safechoices number (from 0 to 10) of choices in the risk assessment where the subject chose the less risky option. round number (from 0 to 20) of the current round of the experiment. riskconsist indicator equal to one if choices in the risk assessment are consistent with transitivity, zero otherwise. gender indicator equal to one if the player is a male, zero otherwise. major indicator equal to one if the player field of study is Finance, Accounting or Economics, zero otherwise. 18 Table 2: Treatment Summary Demand States Unit Cost Parameters Treatment Sessions Subjects Production Opportunities t=1 t = 2 (Low) t = 2 (High) Production Storage Single Production Double Production 4 4 52 48 1 2 {40, 60} {40, 60} {30, 50} {30, 50} {50, 70} {50, 70} 1 1 0.25 0.25 Table 3 shows ex ante expected equilibrium choices by treatment. We derive expected equilibrium choices in Appendix A with the use of the average risk aversion that is elicited with the use of the Holt and Laury (2002) lottery choice procedure. Equilibrium choices within the experiment can differ from reported values due to the influence of the random demand variable and the subgame-path of the current game. We report equilibrium values for second-period choices given the subgame-path in squared brackets. Table 3: Expected equilibrium values and summary statistics The table presents expected equilibrium values in t = 0 and summary statistics. Expected equilibrium values are calculated using the average risk aversion of our participants elicited using a lottery choice procedure from Holt and Laury (2002). The median number of “safe” choices is six, which is consistent with α ≈ 0.15. Variable definitions can be found in Table 1. We present equilibrium values and summary statistics for Single Production and for Double Production, separately. Expected equilibrium values given participants’ subgame-path are reported in squared brackets. In addition, we present equilibrium choices for the Single Production setting under risk neutrality/certainty. Standard deviations are reported in parentheses. Single Production Expected equilibrium Summary statistics (α = 0.15) (α = 0) (20 rounds) (Rounds 15-20) quantity 6.47 32.58 34.742 (0.835) 34.178 (0.921) quantity2 Double Production Expected equilibrium Summary statistics (α = 0.15) (α = 0) (20 rounds) (Rounds 15-20) 23.89 16.33 25.755 (0.852) 24.086 (1.024) 16.33 [9.20] 16.33 [9.20] 15.744 (0.919) 13.162 (1.164) spot sales1 3.91 [20.07] 16.34 [20.07] 20.494 (0.804) 19.630 (0.871) 23.89 [18.63] 16.33 [18.63] 17.668 (0.729) 17.556 (0.767) spot sales2 2.56 [14.67] 16.24 [14.67] 14.25 (0.256) 14.55 (0.408) 16.33 [16.33] 16.33 [16.33] 23.83 (0.466) 19.69 (0.736) hedging1 3.80 0.05 15.932 (0.916) 15.069 (1.136) 22.42 0 18.263 (1.295) 17.546 (1.584) hedging2 2.56 [14.67] [-] 16.808 (0.897) 15.599 (0.985) 16.33 [16.33] [-] 17.294 (1.010) 16.180 (1.118) price1 42.18 17.32 17.34 44.88 17.52 17.34 17.34 profit 272.71 530.89 11.681 (0.898) 22.122 (1.061) 354.717 (19.801) 2.22 price2 10.497 (0.588) 22.071 (0.748) 347.731 (8.763) 295.98 533.66 15.640 (0.532) 11.237 (0.425) 347.308 (19.689) 15.043 (0.763) 14.853 (0.959) 386.088 (34.413) 19 The equilibrium values emphasize our hypotheses of an underhedge in the first period (hedging1 < expected spot sales1) and a full hedge in the second period (Single Production: hedging2 = quantity - spot sales1; Double Production: hedging2 = quantity2). The impact of the hedging opportunity on competition is highlighted by the differences in quantities between the first and second period of Double Production. Finally, Double Production significantly increases competition, and we observe large expected sales in t = 1 and consequently a very low price, which is explained by the strategic aspect of the hedging opportunity: Firms compete via the forward and the spot market, thereby yielding low prices. For the second period, the additional production decision allows firms to decide on the market supply in t = 2 in a certainty-like situation—again increasing competition. We also report equilibrium values for risk-neutral decision makers. The difference between riskneutral and risk-averse decision makers is quite large in our setting due to the large variance of demand uncertainty. Note that the case of risk-neutral decision makers (αA = 0) is equivalent to the case under certainty. Thus, in this case the variance of demand uncertainty does not influence the equilibrium. We decided to use a large variance in our experimental setup in order to generate a noticeable effect on participants’ payoffs. Also note that in the Single Production model, in equilibrium firms also make use of the hedging device in the first period in the case of risk-neutral decision makers due to the strategic application of the hedging device. Absent a risk management motive, the hedging decision in this case only reflects the strategic signal to competitors. In the second period, the hedging device can be used only for risk management purposes; thus, risk-neutral decision makers are indifferent with respect to their hedging2 choice. We show the risk-neutral case in the Double Production model only for completeness. In this setting, the decision problem collapses to a corner solution and yields the repeated single-shot duopoly. 5 Experimental Design Both treatments—Single Production (Section 4.1) and Double Production (Section 4.2)—were implemented using z-Tree software (Fischbacher, 2007).13 Treatments were varied between subjects. 13 This study is registered in the AEA RCT Registry (unique identifying number: AEARCTR-0003940), and full experimental instructions are available at http://www.socialscienceregistry.org/trials/3940. The experimental data 20 Table 2 summarizes the treatments. All sessions were run at Durham University in Autumn 2015. Subjects were recruited using ORSEE (Greiner, 2015). Eight sessions were conducted in total, each with an even number of subjects (between 10 and 16), for a total of 100 subjects. On average, subjects earned approximately £14 each. Sessions lasted approximately 1 hour 45 minutes, on average. In both treatments, subjects were randomly and anonymously matched into pairs of two at the start of each of 20 rounds (Strangers matching).14 At the end of the experiment, two of these 20 rounds were randomly selected for payment. We use random, anonymous re-matching across rounds to allow subjects to learn about the decision environment while minimizing the possibility of collusive strategies or other repeated game effects, as is common practice in many laboratory experiments (see, e.g. Andreoni and Croson, 2008; Ferreira et al., 2016). While there is a positive probability of random re-matching with a previous rival, subjects do not see any identifiers of rivals on which to condition behavior. As our equilibrium benchmarks and hypotheses are based on one-shot interaction in each round, this feature is important for our design.15 Following the design of the decision problem, in the Single Production treatment, each round was divided into three stages. In the first stage, each subject chose a production quantity and a forward market position. In addition to their own choices, subject could make guesses about the choices of the other player. Since the game is complex, we provided a projection calculator tool to help subjects understand the game structure and incentives.16 Projections of the calculator tool were based on own-production and the hedging choices of subjects and on guesses that the subjects made about the same decision variables that wouldbe chosen by the other player. Based on these choices and guesses, subjects were shown projections of expected spot and forward prices and profits and standard deviations of profits. Subjects could adjust their choices and guesses using sliders to see how projected prices, profits, and standard deviations changed. Figure 4 shows a screenshot of the decision screen and the calculator tool. supporting this study are available from the corresponding author upon request. Due to a computer crash, one session had only 19 rounds. 15 As will be discussed in our regression results in the next section, we find no significant correlation between current decisions and lagged feedback on the decisions of previous rival players. 16 This approach has been used to aid subject understanding in many other experimental studies of complicated strategic environments (e.g. Durham et al., 2004; Healy, 2006; McIntosh et al., 2007; Gächter and Thöni, 2010; Van Essen, 2012; Van Essen et al., 2012). 14 21 Figure 4: The screenshot shows the decision screen and calculator tool. Subjects can enter their choices and guesses about the choices of the other player and are shown projections of expected prices and profits, as well as standard deviations of profits. In the second stage, each subject learned the outcome of the first stage, and then chose spot market sales and another forward market position. Subjects could again make guesses about the choices of the other player in the second stage and study projections of expected prices and profits, as well as standard deviations of profits, before finalizing their choices. In the third stage, subjects learned the final outcome of the round, including profits earned (denominated in pence) based on the decisions made and realized demand, as shown in Equations (2) and (3). In the Double Production treatment, each round was also divided into three stages. The first stage was similar to the first stage of the Single Production treatment. However, in the second stage of the Double Production treatment, subjects chose an additional production quantity in addition to spot market sales and a forward market position. Subjects could also make guesses about the additional production, spot-market sales, and forward market position of the other player. As in the Single Production treatment, subjects had a calculator tool that showed projections of expected prices and profits and standard deviations of profits based on their choices and guesses about the other player in the first and second stages. In the third stage, similar to the Single Production 22 treatment, subjects learned the final outcome of the round, including profits earned, as shown in Equations (2) and (6). At the end of the experiment, subjects learned which two rounds were selected randomly for payment. Subjects’ risk preferences were elicited using a lottery choice procedure from Holt and Laury (2002). Subjects also answered a brief demographic survey. At the end of the session, each subject was paid privately in cash their profits from the two randomly-selected rounds and the risk elicitation task. 6 Experimental Results To test our hypotheses, we run a number of paired block-bootstrap t-tests on sales, hedging, and production decisions in the low and high demand states.17 Furthermore, we use a multilevel panel model to regress the variable of interest—spot sales1, first-period hedging, second-period hedging— on variables that should matter based on our predictions: risk aversion, the opportunity for additional production, and the state demand. We provide results for the data that were collected from the 20 rounds, and also the results from rounds 15-20 when subjects have gained some experience. Figure 5 shows the path of average production and sales decisions while Figure 6 shows the path of average hedging decisions in the first and second period. Overall, average decisions appear to be fairly stable across rounds. Some decision variables show a trend in early rounds—such as secondperiod production in Double Production and first-period hedging in Single Production. However, these decisions appear to stabilize in later rounds. In particular, the generally stable path of average production is similar to the results that are found in Schubert (2015). Finally, Figure 7 shows the path of average spot and forward prices across rounds. As intended by the model setup, forward and spot prices for the same period are largely consistent and statistically indistinguishable. Moreover, first-period spot and forward prices are below the standard Cournot level, which indicates stronger competition. Double Production prices are slightly higher than Single Production prices in the first period. Second-period spot and forward prices indicate some learning 17 To account for correlation between observations within a session, we use block-bootstrap t-tests, re-sampling at the session level (Fréchette, 2012; Moffatt, 2016). 23 24 Production decision 10 15 20 25 30 0 10 20 30 40 50 0 0 Treatment 5 5 Double production Single production (b) First-period sales decisions Treatment Round 10 15 15 Double production: second period (a) Production decisions Double production: first period Round 10 Single production 20 20 Figure 5: Evolution of production and first-period sales decision over rounds (with 95% confidence intervals). Sales decision in t=1 25 Hedging decision in t=0 Hedging decision in t=1 0 0 5 5 Double production Single production Double production Single production (b) Second-period hedging decisions Treatment Round 10 (a) First-period hedging decisions Treatment Round 10 15 15 20 20 Figure 6: Evolution of hedging decision over rounds (with 95% confidence intervals). 10 15 20 25 30 10 15 20 25 30 26 Price Price 0 10 20 30 40 0 10 20 30 40 0 0 Round 10 Single production: second period forward price Single production: second period spot price 15 (b) Second-period spot and forward prices Double production: second period forward price Double production: second period spot price 5 15 Single production: first period forward price Single production: first period spot price (a) First-period spot and forward prices Double production: first period forward price Double production: first period spot price Round 10 Figure 7: Evolution of prices over rounds (with 95% confidence intervals). Treatment Treatment 5 20 20 by participants—specifically for Double Production. Here, prices slowly increase over rounds and converge to the price levels of Single Production in later rounds. Summary statistics are reported in Table 3. We capture the data for each player and each round, and then take the averages across players for Single Production and for Double Production, separately. Standard deviations of these averages within each round across player are low, which suggests that there is consistency in players’ decisions. Almost 53% of players were in finance and economics, and 32% of players were male (demographic summary statistics are omitted from Table 3). We do not observe a significant difference in the behavior of players by gender or by course of study. Comparing the summary statistics with equilibrium choices, we note that especially in the Single Production model, choices are much larger than anticipated. More precisely, average choices do not seem to be consistent with the risk aversion that is elicited from the Holt and Laury (2002) procedure. Instead, average choices correspond more with the risk-neutrality case (or, equivalently, the case under certainty). This may be explained by a combination of two effects: First, decision makers may weight the demand uncertainty less than appropriate for the given risk aversion or simply underestimate it. In line with this argument, we do not observe statistically significant correlations between subjects’ degree of risk aversion and their hedging decisions (see Tables 4 and 5). Second, decision makers may pay less attention to the strategic implications of the hedging decision than is predicted by the model. The higher hedging choice also mitigates the impact of the demand uncertainty, and yields higher production. Given the participants’ first-period choices, their spot sales1 decisions are in line with the equilibrium values for the given subgame-path. Double Production averages are in the neighborhood of expected equilibrium choices for risk aversion. t-tests indicate that the average choices for production and second-period hedging decisions— given the hedging decisions that were made in t = 1—are not statistically different from the expected equilibrium choices. However, spot sales1 and first-period hedging decisions are statistically different from the expected equilibrium choices. Spot sales1 are again close to equilibrium values for the relevant subgame-path, given the first-period choices. We first study decisions in time t = 1: We begin with the spot sales1 decision. According to our Hypotheses 1a and 1b, subjects decide on spot sales1 to smooth marginal revenues over periods. This reflects the risk-mitigating impact of hedging, as subjects should be less sensitive to price 27 changes given that the forward price is fixed. To test this prediction, we compute the marginal revenues that are implied by the subjects’ decisions in both periods. To reduce the likelihood that the results reflect the subjects’ unfamiliarity with the strategic forces that they face, we provide results for all 20 rounds and also for rounds 15-20. We cannot reject the null hypothesis that the marginal revenues are equal in both periods in high demand realizations. However, when demand is low, subjects choose to sell in a way such that the marginal revenues of the first period are significantly higher than the marginal revenues of the second period. The explanation is that production was too high for the low state demand. Therefore, subjects made their decisions such that the first-period marginal revenue was zero—which we cannot reject from a one tailed t-test (p = 0.978)—and then sold the remaining quantity in the second period at (possibly) negative marginal revenue. Results from regressing spot sales1 on theoretically important variables are reported in Table 4.18 We regress spot sales1 on the players’ own quantity produced and own hedging in the first period as well as on the quantity produced and the hedging of their competitors. Consistent with Hypotheses 2a and 2b, “safechoices” is not significantly correlated with spot sales1, in either model. To study Hypotheses 3a and 3b and the strategic impact of the hedging choice on sales, we examine whether spot sales1 increase with players’ own hedging and decrease with their opponent’s hedging. Own first-period hedging is significantly positive, but only in Single Production. Moreover, the coefficient is much smaller than the expected value (+1/3). Also, the results in Table 4 suggest that the coefficients on competitors’ hedging are not significant. Finally, according to Hypothesis 6, sales decisions should be significantly larger for Double Production. Our results support this hypothesis. With respect to the hedging decisions in t = 1: According to Hypotheses 4a and 4b, subjects should employ a full hedge in the second period to ensure deterministic profits in the final stage. To test this prediction, we examine the statistical significance of the difference between the second-period hedging (set in t = 1) and the quantity that is available to sell in the final stage. The results for the 18 We use multilevel Tobit models to account for the bounds on the decision variables (between 0 and 50) with random effects at the individual and session levels to control for correlation within individuals and within sessions (Fréchette, 2012; Moffatt, 2016). To examine whether current decisions are correlated with previously observed feedback, we also ran models that are similar to Models (1) and (2) in Tables 4, 5, and 6 including controls for the lagged decisions of the other player in the previous round. We found no significant correlations, and chose to omit these results for brevity. We also tried alternative specifications using the log of round or round fixed effects, and we found very similar results to those presented in Tables 4, 5, and 6. 28 Table 4: Spot Sales1 Multilevel Tobit Regressions Models 1-2 report the results from all 20 rounds, whereas Models 3-4 report the results from only rounds 15-20. Models 1 and 3 are restricted to the Single Production model, and Models 2 and 4 are restricted to the Double Production model. Variable definitions can be found in Table 1. All models include random effects at the individual and session levels. (1) (2) (3) (4) quantity 0.523∗∗∗ (0.0287) 0.547∗∗∗ (0.0259) 0.473∗∗∗ (0.0526) 0.559∗∗∗ (0.0424) otherquantity -0.0530∗ (0.0230) 0.0128 (0.0216) 0.0511 (0.0394) -0.00653 (0.0328) hedging1 0.0799∗∗∗ (0.0241) 0.00666 (0.0210) 0.0957∗ (0.0408) 0.0215 (0.0334) otherhedging1 0.00958 (0.0198) 0.0230 (0.0163) 0.0217 (0.0315) 0.0250 (0.0234) highstate 2.616∗∗∗ (0.409) 2.281∗∗∗ (0.392) 1.663∗∗ (0.633) 2.743∗∗∗ (0.582) safechoices -0.261 (0.344) -0.0608 (0.210) -0.673 (0.365) -0.218 (0.231) round -0.0707∗ (0.0354) 0.131∗∗∗ (0.0346) -0.195 (0.175) 0.252 (0.157) riskconsist 1.229 (1.430) -1.116 (1.184) 1.298 (1.511) -0.742 (1.312) gender -0.143 (1.152) -0.395 (1.111) 0.440 (1.222) 0.309 (1.219) major -1.160 (1.118) 0.912 (0.993) 0.235 (1.173) 0.741 (1.105) constant 3.311 (2.584) 1030 0.808 (2.008) 960 5.364 (4.591) 302 -1.168 (3.558) 288 Observations Standard errors in parentheses. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 29 Table 5: Second-Period Hedging Multilevel Tobit Regressions Models 1-2 report the results from all 20 rounds, whereas Models 3-4 report the results from only rounds 15-20. Models 1 and 3 are restricted to the Single Production model, and Models 2 and 4 are restricted to the Double Production model. Variable definitions can be found in Table 1. All models include random effects at the individual and session levels. quantity (1) (2) (3) (4) 0.232∗∗∗ (0.0498) 0.0434 (0.0436) 0.156 (0.0830) -0.000503 (0.0777) quantity2 0.147∗∗∗ (0.0351) 0.298∗∗∗ (0.0686) otherquantity 0.0816∗ (0.0406) -0.00593 (0.0353) 0.0942 (0.0634) 0.0747 (0.0586) otherhedging1 -0.0141 (0.0348) 0.0217 (0.0268) -0.0982 (0.0507) 0.160∗∗∗ (0.0415) highstate -1.803∗ (0.719) -1.599∗ (0.667) -1.115 (1.016) -2.587∗ (1.101) safechoices -0.750 (0.684) 0.493 (0.465) -0.311 (0.725) 0.592 (0.507) round -0.183∗∗ (0.0617) -0.0688 (0.0583) -0.542 (0.279) -0.573∗ (0.279) riskconsist 0.0653 (2.839) -2.572 (2.617) -0.316 (2.849) -3.387 (2.839) gender -4.767∗ (2.289) 1.533 (2.469) -4.967∗ (2.343) -0.373 (2.717) major -5.478∗ (2.231) 4.575∗ (2.196) -4.953∗ (2.266) 6.030∗ (2.403) constant 16.14∗∗ (4.930) 1030 10.85∗∗ (4.093) 960 23.48∗∗ (7.733) 302 14.18∗ (6.795) 288 Observations Standard errors in parentheses. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 30 Table 6: First-Period Hedging Multilevel Tobit Regressions Models 1-2 report the results from all 20 rounds, whereas Models 3-4 report the results from only rounds 15-20. Models 1 and 3 are restricted to the Single Production model, and Models 2 and 4 are restricted to the Double Production model. Variable definitions can be found in Table 1. All models include random effects at the individual and session levels. (1) (2) (3) (4) quantity 0.267∗∗∗ (0.0412) -0.170∗∗∗ (0.0435) 0.438∗∗∗ (0.0791) -0.336∗∗∗ (0.0733) safechoices 0.254 (0.655) 0.788 (0.739) 0.481 (0.799) 1.086 (0.913) round -0.163∗∗ (0.0502) -0.175∗∗ (0.0562) -0.0933 (0.251) 0.154 (0.239) riskconsist -3.795 (2.717) -5.583 (4.159) -4.569 (3.322) -6.644 (5.123) gender -6.338∗∗ (2.186) -0.338 (3.923) -5.736∗ (2.677) 0.106 (4.848) major 0.311 (2.127) 4.019 (3.489) -0.133 (2.592) 5.090 (4.307) constant 11.47∗∗ (4.357) 21.83∗∗∗ (5.984) 3.996 (7.131) 18.56∗ (8.485) 1030 960 302 288 Observations Standard errors in parentheses. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 31 full sample suggest that there is an under hedge, as the difference is statistically negative. When we consider rounds 15-20, however, the results suggest that we cannot reject the null hypothesis: subjects tend to hedge fully for the Single Production setting (p-value is 0.486), but we still reject the null hypothesis for the Double Production setting. In our regression analysis, we test whether the second-period hedging decision is independent of the risk aversion of the players. The results that are reported in Table 5 suggest that own-hedging in the second period is indeed independent of the risk aversion of subjects—as expected. Moreover, the table shows that, naturally, the hedging amount increases with the quantities that are produced in the two periods and decreases with the demand realization of the first period (since firms sell more of their first-period production on the spot market in this case). Our results also indicate that male decision makers hedge less in the Single production model. Next, we turn to first-period hedging decisions: According to Hypotheses 5a and 5b, the initial hedging decision should be an underhedge. This means that we should observe an initial hedging decision that is smaller than expected sales. To test the hypotheses, we calculate the expected sales of participants that would result in equilibrium based on their production quantity and initial hedging choice as well as their guesses as to their competitors’ choices. Then, we compare the expected sales with their hedging decision. For Single Production, average first-period hedging (15.93, Table 3) is significantly smaller than expected spot sales1 (19.61; t-statistic of 9.73). Similarly, average first-period hedging in Double Production (18.26, Table 3) is significantly smaller than expected spot sales1 (19.42; t-statistic of 2.64). Thus, we observe a significant underhedge, which suggests that players do take into account the strategic impact of their first-period hedging decision. Finally, we provide regression analyses on the own first-period hedging. Surprisingly, the results that are reported in Table 6 suggest that own-hedging in the first period is not correlated with the risk-aversion coefficients, even when we consider rounds 15-20 only. Similar to the second-period hedging choice, we observe a tendency of males to hedge less than females—though this difference is not always statistically significant. Lastly, we find that individuals reduce their first-period hedging decisions in later rounds, on average, as indicated by the negative coefficients on round in Models 1 and 2. This finding supports the notion of a small trend in early rounds observed in Figure 6. 32 7 Conclusion In this paper, we experimentally investigate the influence of a simultaneous hedging opportunity on firm choices and competition in incomplete markets. In markets with imperfect competition, the risk management hedging opportunity introduces a strategic dilemma for competitors: Corporate hedging increases the competitiveness in the market; but the hedging opportunity also allows producers to deal with their risk exposure. Consequently, firms must weigh the advantages of being able to hedge their risk exposure against the disadvantages of more intense competition. We consider a multi-period duopoly under demand uncertainty with a hedging opportunity and the possibility of storage in a simple framework with (µ, σ)-preferences. Our setting yields the following hypotheses: Duopoly firms consider the impact of their hedging choice on the market equilibrium and are thus willing to take a risky position to mitigate the adverse effect of the hedging actions on market prices. The simultaneous hedging opportunity should increase competition. Our experimental results support the hypotheses: We find that subjects appear to be aware of the strategic effect of the hedging device, and thus reduce their own first-period hedging to an underhedge and adjust their spot sales in t = 1 according to their hedging choice. However, we do not find evidence that subjects account for their competitors’ financial decisions when making their own quantity supply decisions. Moreover, as the game is closer to a simple repetition of the single-shot game in Double Production, competition increases to a level that completely negates duopoly profits. Hence, we find strong evidence that the hedging opportunity significantly increases competition—even in a simultaneous setting. These results supplement the literature that provides evidence of a strategic dilemma that is introduced by a hedging opportunity in a sequential setting (Allaz, 1992; Allaz and Vila, 1993). We also supplement the findings by Brandts et al. (2008) who document that, in absence of risk and a strategic dilemma, the introduction of a forward market significantly increases competition in the market. Moreover, our results are related to the observations of Mueller (2006) who finds that the behavior of players in a Cournot duopoly with multiple production periods does not differ significantly from standard one-period markets—in contrast to theoretical predictions. In our Double Production treatment, first-period choices are significantly larger than in the one-period interaction—due to 33 the strategic effect of the hedging opportunity. Acknowledgements This work was supported by the DUBS Seedcorn Fund from Durham University. We are grateful for comments from editor Lawrence J. White and two anonymous referees, as well as Udo Broll, Jack Wahl, Bernhard Ganglmair, and participants of the 2014 Cambridge Business & Economics Conference, the Experimental Finance North American Finance Meeting 2016, and the 2017 AEA Annual Meeting in Chicago. A previous version of this paper was circulated under the title “Strategic corporate hedging”. We thank Minh-Lý Liêu and Sonja Warkulat for outstanding research support. Matthias Pelster gratefully acknowledges the warm hospitality of the Durham University Business School during his visits. Any errors, misrepresentations, and omissions are our own. A Expected Equilibrium Values: Numerical Example This appendix derives the equilibrium values that are shown in Table 3. A.1 Single Production setting We begin with the Single Production setting (see Section 4.1). First, we derive second-period choices. We present mathematical representations for firm A. As the duopoly is symmetric, similar representations apply for firm B. At this point in the model, all decisions from the first period, the realization of the demand uncertainty of the first period, and the forward rate for the second period are common knowledge. Thus, firms maximize A B A ΦA L = (50 − (s1 + s1 ))s1 − {z } | revenues from sales in t=1 q1A |{z} fixed production costs B A A + ȟA (f (0, 1) − (50 − (sA 1 + s1 )) − 0.25(q̌1 − s1 ) |1 {z } | {z } profits from t=0 hedging position αA storage costs A A A 2 + {(50 − (q̌1A + q̌1B − sA − sB 200 (q̌1A − sA 1 ))(q̌1 − s1 ) − 1 − h2 ) } (13) | {z1 } 2 revenues from sales in t=2 34 A by choosing sA 1 and h2 . The first-order conditions are B A A B A B A A A A 50 − (2sA 1 + s1 ) + ȟ1 + 0.25 + {−50 + (2q̌1 + q̌1 − 2s1 − s1 ) + α 200 (q̌1 − s1 − h2 )} = 0 (14) and A αA 200 (q̌1A − sA 1 − h2 ) = 0. (15) Subtracting these yields Equation (4). The optimal supply decision in t = 1 fulfills (E[ε1 ] = 50) B A A A B B 50 − (2sA 1 + s1 ) + ȟ1 = 50 − (2q̌1 − 2s1 + q̌1 − s1 ) − 0.25. (4 revisited) For the second-period hedging decision, the full hedge immediately follows from Equation (15): A A hA 2 = q̌1 − s1 . (16) Equation (4) yields a system of two equations (firm A and firm B) with six unknowns. Solving for spot-sale decisions, this leads to sA 1 = 1 1 1 B 1 + q̌1A + hA 1 − h1 . 24 2 3 6 (17) Taking these future decisions into account, decision makers initially maximize A B A ΦA L = (50 − ŝ1 − ŝ1 ) · ŝ1 − | {z } revenues from sales in t=1 q1A |{z} production costs αA A 2 − 0.25(q1A − ŝA 100(ŝA 1 )− 1 − h1 ) 2 | {z } storage costs αA A 2 A 2 B A A + {(50 − (q1A + q1B − ŝA (200(q1A − ŝA 1 − ĥ2 ) + 100(ĥ2 ) )}. 1 − ŝ1 ))(q1 − ŝ1 ) − | {z } 2 {z } | revenues from sales in t=2 =0, full hedge based on the expectations in time t = 0. Simplified first-order conditions that take second-period choices (that are made in t = 1) into account are 48.875 − q1A qB − 1 − 100 · αA · 2  35 3 A 1 2 hB 1 q1 − − hA 1 + 4 48 3 12  =0 (18) and 2 · 100 A ·α · 3  1 qA 2 hB 1 + 1 − hA 1 − 24 2 3 6  + 1 2 1 B − hA h = 0. 1 − 72 9 18 1 (19) This system of four equations (similar equations apply for firm B) with four unknowns (considering that risk aversion is a fixed parameter) yields expected equilibrium choices in t = 0. For αA = 0.15, we obtain q1A = q1B = 6.47 and B hA 1 = h1 = 3.80. B Equation (17) yields resulting spot sales1 in t = 1, sA 1 = s1 = 3.91. Based on the spot-sale decisions, the equilibrium forward price is 1 1 1 B f (0, 1) = 50 − (q1A + q1B ) − (hA · 0.25 = 42.18. 1 + h1 ) − 2 6 3 Expected prices are given by the inverse demand function, B p1 = 50 − (sA 1 + s1 ) = 42.18 and B p2 = 50 − (q1A + q1B − sA 1 − s1 ) = 44.88 Finally, expected profits read A A A A E[π̃1A ] = E[p̃1 ]sA 1 − q1 + h1 (f (0, 1) − E[p̃1 ]) − 0.25(q1 − s1 ) = 42.18 · 3.91 − 6.47 + 0 − 0.25 · (6.47 − 3.91) and A ˜ E[π̃2A ] = E[p2 ](q1A − sA 1 ) + h2 (f (1, 2) − E[p̃2 ]) = 44.88 · (6.47 − 3.91) + 0 and consequently E[π̃ A ] = E[π̃1A ] + E[π̃2A ] = 272.71. 36 (5 revisited) For risk-neutral decision makers (αA = 0), the system of equations (18) and (19) simplifies to q1A = 48.875 − q1B 2 and hA 1 = 1 1 − hB , 16 4 1 q1A = 2 · 48.875 3 which yields and hA 1 = 3 . 60 Second-period decisions, prices, and profits are obtained in the same manner as above. A.2 Double Production setting Next, we turn to the equilibrium values in the Double Production setting (see Section 4.2). We again start with the second-period decision problem. Firms maximize ΦA = p(Q1 )sA | {z 1} revenues from sales in t=1 − q̌1A |{z} first-period production costs + {p̄(Q2 )(q2A + q̌1A − sA 1 )− | {z } revenues from sales in t=2 B A A + ȟA (f (0, 1) − (50 − (sA 1 + s1 ))) − 0.25(q̌1 − s1 ) |1 {z } | {z } q2A |{z} profits from t=0 hedging decision − αA 2 storage costs A 2 200 (q2A + q̌1A − sA 1 − h2 ) }, second-period production costs where Q1 and Q2 denote the industry supply in t = 1 and t = 2, respectively. To obtain the interior solution, we derive the first-order conditions (ε̄1 = 50): B A A B A B A B 50 − (2sA 1 + s1 ) + ȟ1 + 0.25 − (50 − (2q2 + q2 + 2q̌1 + q̌1 − 2s1 − s1 ) A − αA 200 (q2A + q̌1A − sA 1 − h2 )) = 0, (20) B A A A A A 50 − (2q2A + q2B + 2q̌1A + q̌1B − 2sA 1 − s1 ) − 1 − α 200 (q2 + q̌1 − s1 − h2 ) = 0 37 (21) and A αA 200 (q2A + q̌1A − sA 1 − h2 ) = 0. (22) The second-period hedging decision immediately follows from Equation (22). The addition of Equations (21) and (22) yields Equation (9): marginal costs = marginal revenues. Thus, firms sell 49/3 units (= standard Cournot quantity) in the second period and adjust their second-period production accordingly. Also, we obtain Equation (10): B A 50 − (2sA 1 + s1 ) + ȟ1 = 1 − 0.25 = 0.75. (10 revisited) Solving for the equilibrium, we obtain sA 1 = 50 − 0.75 2 A 1 B + ȟ1 − ȟ1 ≤ q1A . 3 3 3 (23) Then, decision makers initially maximize B A ΦA = (50 − (ŝA 1 + ŝ1 ))ŝ1 − | {z } revenues from sales in t=1 production costs prof |{z} + q1A |{z} αA A 2 − − 0.25(q1A − ŝA ) 100 (ŝA 1 1 − h1 ) | {z } 2 storage costs (q1A − ŝA ) | {z 1 } + net revenues from sales in t=2 − αA 2 100 (q1A − ŝA 1) . 2 non-incurred second-period production costs Simplified first-order conditions to determine the interior solution, considering the second-period choices, are q1A − 0.25 49.25 2 A 1 B − h1 + h1 + =0 3 3 3 100 αA and αA 100  2 A 49.25 5 A 1 B q − − h1 + h1 3 1 9 9 9  + 49.25 4 A 1 B − h1 − h1 = 0. 9 9 9 This system of four equations with four unknowns (again considering that risk aversion is a fixed parameter) yields expected equilibrium choices in t = 0. For αA = 0.15, we obtain q1A = q1B = 23.89 B and hA 1 = h1 = 22.42. 38 As a result, spot sales1 amount to sA 1 = 23.89 (see Equation (23)). With nothing in storage, the firms produce q2A = q2B = 16.33 and hedge the entire production on the second-period forward B market hA 2 = h2 = 16.33. Prices and firm profits follow immediately from the inverse demand function and Equations 7 and 8. For risk-neutral decision makers (αA = 0), the decision problem collapses to a repeated single-shot duopoly under certainty with a corner solution, which is consistent with the notion of Broll et al. (2011) that strategic considerations are absent in a setting where firms decide on their production and hedging decision at the same time. References Adam TR, Frenando CS, Golubeva E (2015) Managerial overconfidence and corporate risk management. Journal of Banking and Finance 60:195–208 Allaz B (1992) Oligopoly, uncertainty and strategic forward transactions. 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W1994051516.txt

C. D. lOO CHILD. [VOL. XX. T H E ARC IN A MAGNETIC F I E L D . BY. C. D. CHILD. T H E following is an account of certain phenomena observed when the electric arc is placed in a magnetic field. A preliminary description of these were given before the American Physical Society in March, 1904, and an abstract of the work appeared in the PHYSICAL REVIEW. 1 The First Experiment.— The connections of the first experiment are shown in Fig. 1. A and B are two carbons 12 mm. in diameter. c and d are carbon pencils 3.1 mm. in diameter placed near to the side of the arc. These are connected through a millivoltmeter having a resistance of 177 ohms. This voltmeter was quite dead beat and the period was about 1 sec. An electromagnet is placed near the arc and produces a field normal to the plane of the figure. It was found quite impossible to place c and ) dy so that the potential difference between them continued to be zero, or even to place them so that it was constant. The pointer of the voltmeter would at times remain quite for Fig. 1. several seconds and then change suddenly, sometimes changing only a slight amount and at other times reversing. It is impossible, at least, in air to keep the potential difference constant, because c and d are consumed rapidly and before the arc reaches a quiescent condition they are burnt away from it. But even though the reading of the voltmeter was not constant, it was evident that whenever there was a sudden change in the magnetic field, there was a corresponding change in the reading of the voltmeter. If the direction of the field was such that the arc was € 1 PHYS. REV., 18, 370. No. 2.] THE ARC IN A MAGNETIC FIELD. IOI displaced to the right, d would become positive, as compared with its former condition, and c negative; if to the left, c would be positive, and d negative. If in the first example d was already positive, it would become more so, and if negative, it would become less negative. Electrometer Substituted for Voltmeter.— It was soon found that a voltmeter was not an ideal instrument for the purpose. As has been stated its resistance was only 177 ohms and the apparent resistance between the two pencils in the arc, even when they were very hot, appeared to vary from a few ohms to several hundred. On the other hand the period of such electrometers as were at hand was so great that the condition of the arc changed much during a reading. As has been stated, the rapid burning of the electrodes and especially the exploring pencils made it desirable to take readings as rapidly as possible. Moreover a voltmeter is a much more convenient instrument to use than an electrometer. As a result the experiments were nearly all performed first with a voltmeter and more careful measurements were afterwards taken with an electrometer. The electrometer used had a period of 16 seconds and was very nearly dead beat. Thus the experiment already described was repeated using an electrometer instead of a voltmeter, and the same results were observed as before. Modifications of this Experiment. — As will be seen later there is doubt about the real significance of this phenomenon, and consequently many variations of the experiment were tried. One of these may be described as follows : It has been shown that d in Fig. 1 has a higher potential when the arc is deflected by a magnet to the right and a lower one when it is deflected to the left. If A is the positive carbon, then the potential difference between A and d should decrease when the arc is shifted to the right, and increase when shifted to the left. Such changes were, indeed, found to occur. On the contrary when the voltmeter was connected between B and d, the readings increased on deflecting the arc to the right, and decreased on deflecting it to the left. The opposite results followed when the exploring pencil was placed at c instead of at d. A slight modification of this experiment was made by connect- 102 a v. CHILD. [VOL. XX. ing a shunt between A and B and connecting d to some point of this shunt. The observation again indicated that the potential of d is increased when the arc is deflected to the right, and diminished when it is deflected to the left. The electric force through the arc was also measured both with and without the magnetic field, and no difference could be detected in the two cases. Rapid Reversing. — Since the potential difference between c and d was not for any length of time constant, an arrangement was used by which the ordinary fluctuations of this potential were eliminated and those produced by the magnetic field were retained. To do this the connections between c and d and the voltmeter were rapidly reversed. The voltmeter would thus normally show no deflection. But at the same time that these connections were reversed those running to the electromagnet were also reversed. The effect due to the magnetic field was thus shown as before. The reversals were made by a wheel, as shown in Fig. 2. This was made of fiber, and was 10 cm. in diameter. It i *s a combination of a commutator and a collecting J anc S* 2 are connected to the carbon pencils, 3 and 4 to the voltmeter. With each revolution of the wheel two reversals would occur. A similar arrangement was used for reversing the magnetic field the reversal of one circuit taking place at the same time as that of the other. With this arrangement a reading of the voltmeter was attained which was at least always in the same direction, and the reading was reversed when the direction of the current through the arc was reversed, or the connections to the reversing wheel for the electro-magnet. The readings indicated as before that the current passed from d through the voltmeter to c, when the arc was deflected to the right The arc was then removed and c and d were connected by copper wires. No effect, whatever was then observed. This shows that the effect was not due to any E.M.F. induced by the alternations of the magnetic field. Arc Deflected Mechanically. — But though the accuracy of the first observations was thus proven, there was still the possibility that /^TT\ \iC\\\ \V_J/y \4\ \ . rm No. 2.] THE ARC IN A MAGNETIC FIELD. 103 the magnetic field was not the direct cause of the observed effect, that rather the mechanical motion of the arc was the direct cause. To test this the arc was blown to one side by a blast of air. This usually produced a smaller reading of the voltmeter whatever that reading might be, when that instrument was used. This was due to the fact that when the arc is blown to one side, the resistance between the pencils is increased. When an electrometer was used, it was found that with the arc blown sufficiently to one side, so that one of the pencils became much cooler than the other, the cool one always became charged positively. Further investigation showed that the space about the arc had a higher potential than the arc itself aftd that when the arc was blown away from one of the pencils, that pencil became charged positively, merely because it was assuming the potential of the surrounding space. The difference between the phenomenon in this case and that when the arc was deflected by a magnet was especially noticeable when the arc was entirely displaced from one of the pencils. Such a pencil was charged negatively when the arc was deflected by a magnet, and positively when deflected by a blast of air. Moreover as long as both carbons were kept very hot no regular effect could be detected by blowing the arc, while deflecting it by a magnet always produced an effect. The difference in these results was also strikingly shown when the potential difference between one of the pencils and one of the electrodes was examined. It has already been stated that in certain cases such potential differences increased, while in others they decreased, when the arc was deflected by a magnet. When it was blown to one side the potential difference in every case increased, provided the exploring carbon was kept in that part of the arc where the conductivity was good. Further Test of Effect Produced by Mechanical Motion. — In order to test further the suggestion that the effect here studied was produced by the mechanical motion of the arc and not by the magnetic field, a fan was arranged which blew the arc first in one direction and then in the other. This arrangement is shown in Fig. 3. W is a wheel taking the place of the reversing wheel previously de- io4 C. D. CHILD. [VOL. XX. scribed. P is a pin fastened to the wheel and moving in the slit RH. H is a hinge. At R two fans F and Ff are fastened at R and the arc is placed between them. The reversals of the motion of the fan occurred at the same time as those of the connections to the voltmeter, so that this arrangement produces the same effect as far as mechanical motion is concerned as the magnetic field. But no deflection of the voltmeter was observed even when the velocity of the fan was increased until the arc was blown out. Varying the Rapidity of the Reversals. — The first thing varied was the period of the reversals. This tests again the possibility that the effect is due to the unequal heating of the two carbon pencils. Though the temperature of the carbon of the arc follows very closely the fluctuations of the arc, there was the possibility that the reversals could be produced so rapidly that the effect would decrease. It was found, however, that increasing the rapidity of the reversals did not diminish the effect. Thus when the number of reversals was increased from 4 per second to 25 per second, the reading of the voltmeter diminished from .35 volts to .30 volts. Several observations were made, but this is the average, and further investigation showed that this decrease in the reading is not due to any change in the conditions of the arc, but to the self-induction of the electro-magnet and the voltmeter and to changing contact resistance. Distance Between Exploring Pencils Varied. — B The distance between the exploring carbons was then varied. It was found, however, that the results Fig. 4. obtained when the pencils were more than 7 or 8 mm. apart depended very largely on the height of the exploring carbons. Thus at ah the potential difference was small, at afbfy it was much larger, while at af,bny it was reversed. It was probably small at ab because there they were only barely touched by the arc. At afbf they were more directly in the arc. At anbn the effect appeared to be due to the mechanical motion of the arc, and No. 2.] THE ARC IN A MAGNETIC FIELD. 105 not to any magnetic cause. If the arc is blown to the right bn will be in the hot gases arising from the arc more than an. It will consequently have more nearly the potential of the arc, and as we have seen the arc is negative as compared with the surrounding space. Current through Arc Varied. — T h e current through the arc was varied from 5 to 25 amperes, but no definite change in the potential difference between the exploring pencils was discovered. According to the measurements taken the change was at least less than one part in twenty. Length of Arc Varied. — The effect was found to increase when the length of arc was increased. The following observations were taken, using an electrometer, the alternating apparatus, a current of 12 amperes, and a distance between the pencils of 9 mm. TABLE Length of Arc. 3 6 9 I. Potential Differences Between Pencils. 1.7 2.5 3 Strength of Field Varied. — Next the strength of the field at the arc was varied. To determine the strength of the magnetic field a long solenoid 3.5 sq. cm. in cross section was wound with 13.8 turns per cm. One hundred turns of wire were wound about this and the throw of a ballistic galvanometer was observed when a given current was sent through the primary turns. This was compared with the throw produced by 100 turns at a given distance from the electromagnet which had been used with the arc. The effects observed in this way with the electromagnet at different distances from the arc are given in Table II. TABLE Distance from Solenoid. 6 9 12 15 Magnetic Field. 13.4 7.5 5 3 II. Potential Difference Between Pencils. 4 3 2.2 .8 The distances between the arc and the end of the magnet are given in column 1, the strength of the field in electromagnetic units in column 2, and the potential difference between the exploring car- io6 C. D. CHILD. [VOL. XX. bons in volts in column 3. The current through the arc was 12 amperes and the length of the arc was 5 mm. These readings were taken with the electrometer and alternating apparatus. Phenomenon Dependent on Quality of Carbons Used. — While making these observations it was found that the amount of the effect depended very largely on the kind of carbon used. Those first used were solid, but in changing the apparatus cored carbons were unintentionally substituted, and for some time none of the experiments which had been performed could be repeated. In time it was found that it was the change in carbons which caused the difference. It was found that, in general, solid carbons gave a greater effect than cored. It happened that the first ones used gave a greater effect than any others investigated. I do not know their make, as they had been in the laboratory a number of years. Different Substances in the Arc. — Further investigations showed that the effect was diminished very much when certain salts were placed in the arc. Indeed with some it vanished entirely. In testing this a hole was drilled for 2 or 3 cm. into the carbon and this was filled with the salt to be investigated. Only a very small effect was found with NaCl, and none whatever with KN0 3 . Moreover, no effect was found with the arc between iron or copper terminals. An attempt was made to use graphite terminals, but it was found difficult to maintain any arc with the terminals far enough apart to insert the exploring carbons, even when using a voltage of 200 volts, and when the exploring carbons were inserted a very small magnetic field was sufficient to extinguish the arc. As a result no measurements were made with graphite terminals. Phenomena in a Vacuum.— It was also found that the effect disappeared in a vacuum. The observations were made in a tube which has been described in a previous article l the exploring pencils were 1.6 mm. in diameter. They were led into the tube by the method which had been previously used in examining the electric force in the arc. A series of readings thus taken are given in Table III., the distance between the exploring carbons was 5 mm. and they both became white hot while in the arc. The arc was 7 mm. long. The current was 10 amperes. The end of the electro1 PHYS. R E V . , 19, 117. No. 2.] THE ARC IN A MAGNETIC FIELD. IOJ magnet was 7 cm. from the arc, making the field at the arc 10 electomagnetic units. Column 1 gives the pressure of gas in millimeters of mercury. Column 2 gives the difference in potential between the exploring carbons in volts. TABLE III Potential Difference Between Pencils. 3.3 2.5 1.9 2 1.2 .7 .4 .2 Pressure of Gas. 750 550 400 300 200 100 50 20 At 6 mm. pressure no effect could be detected either with an electrometer or with a voltmeter. A galvanometer having a constant of 3.6 x icr~5 ampere per scale division was also used and it was even then impossible to detect any effect. The galvanometer had a resistance of 445 ohms and a period of 13.5 sec. It seemed useless to try a more sensitive galvanometer. For without any magnetic field the potential differences betwen the two pencils in the arc fluctuated from zero to .2 volt and one cannot well eliminate an effect of this size so completely as to study one which was less than .001 volt. Possible Explanations.— During the experiments it was noticed that a magnetic field has less effect in deflecting the arc to one side when it is in a vacuum than when in air. Thus the field used with the vacuum was 15 units and it scarcely affected the appearance of the arc, while it extinguished it entirely when using the same length of arc and same current in air. It was thought at first that this might possibly be the cause of the disappearance of the effect in a vacuum. But the effect also decreases when NaCl was placed in the arc, and in that case the arc is blown fully as much to one side as with ordinary conditions. In a recent article by Weintraube l it is stated that when a magnetic field is produced about a mercury arc in a vacuum the bright spot at the cathode moved in one direction, while the vapor of the 1 Phil. Mag. (6), 7, 95. io8 C. D. CHILD. [VOL. XX. arc moved in the opposite. Special notice was, therefore, taken of the arc in air and it was found that generally the bright spot did not move at all, when the magnetic field was produced, but that when it did, it moved in the same direction as that of the vapor of the arc. It is evident that an explanation for these phenomena must explain the many cases where the effect disappears. We find that wherever it disappears the drop in potential at the anode is much smaller than it is with carbons where it is large. Thus with a solid carbon the drop in potential between the anode and an exploring pencil placed as near to it as possible was 41 volts. With a cored carbon with which the effect was scarcely noticeable the drop was 30 volts. With the iron with which experiments were performed it was 16 and with copper 21 volts. In a previous article 1 it has been shown that with the carbons there used the drop was 50 volts with a pressure of j$o mm. of mercury, 43.5 at one of 200 mm., and 26.8 at 10 mm. Thus is all cases where the effect disappears the drop in potential at the anode is much diminished. There is another set of facts with which the phenomena may be connected. It was shown in a previous article that with ordinary carbons the positive discharge from an arc to a surrounding cylinder was greater than the negative.2 This indicated that the positive ions had the greater velocity. When salts were placed in the arc, or when other electrodes than carbon were used, the negative discharge was approximately as great, or even greater than the positive. In a more recent article 3 it was shown that in a vacuum the negative discharge again became the greater. So that in all cases which have thus far been studied when the drop in potential at the anode is small, the discharge from an arc to a cylinder becomes relatively larger and the effect due a magnetic field disappears. In view of the complications existing in the arc it may be unwise to offer any explanation of the phenomena here described, and yet an attempt to do so may lead at least to further investigation, and I would, therefore, suggest the following: * P H Y S . R E V . , 19, 120. 2 PHYS. R E V . , 12, 137. 3 PHYS. R E V . , 19, 125. No. 2.] THE ARC IN A MAGNETIC FIELD. 109 The usual equation for the Hall effect is Z = ^ XH' (u — v) where Z equals the electric force due to the Hall effect; X, the impressed electric force ; H> the magnetic field ; and it and v, the velocities of the positive and negative ions respectively for unit electric force. The directions of the quantities are those indicated in Fig. 5. If we should apply these equations to the arc we would conclude that the positive ions have velocities enormously greater than the negative. However, in deriving this equation the assumption is made that the ions have no component of motion / perpendicular to the X direction, whereas in the arc ' H p i e 5 there is such motion. It is the motion of the ions to one side which causes the arc to be deflected. This equation, therefore, can not be said to hold in this case. However, from physical considerations we shall be led to the same general conclusion as when treating the matter mathematically, that is, we are led to believe that the positive ions have the greater velocity. In the arc both positive and negative ions are deflected, but those having the greater velocity will be deflected most. This can be shown as follows: Let e = the charge on an ion. Let m = the mass of the ion. Let ff= the magnetic force in the direction of the Faxis. Let Jf = the electric force in the direction of the X a x i s . Let x and z be the distances from the origin along the X and Z axes to the point where the ion is at a given instant. If the X, Yt and Z axes are at right angles to each other and if the ion starts from a condition of rest at the origin x 1 — / — sin cot) and x X - , — cos; cot) where to = 1 He m x Thomson's Conduction of Electricity through Gases, 88. C. D. I IO CHILD. [VOL. XX. If /j is the time necessary for the ion to move over its mean free path and xx and j \ the corresponding values of x and y, *i 3 9° But the average velocity during this time, if there had been no magnetic force would have been Xetxj 2m. This will be the same as the average velocity for any number of mean free paths and if we let v equal the average velocity when the electric force is one " 2111 Then 1 tot. = I and yx__2Hva xx 3 = 2Hv m (2Hvaf 90 If now va=* 1000 cm. per sec. for one volt per sec. and / / = 10 E.M. units the ratio between the first and second term equals I io~ 9 , so that for such cases as are here considering yjxx = $Hva. There will be 1 jxx of the collisions per cm. so that the total deflection in going 1 cm. in the ^--direction = yx/xx = \Hva which is greater, the greater the average velocity of the ion. This case is evidently quite different from that of cathode rays. There practically all of the deflection takes place while the ion is moving with uniform velocity, while here it is supposed to occur where the velocity is uniformly accelerated. If then the negative ions are deflected most, the part of the arc to the right, namely, in the Z direction, will have an excess of negative ions, and the potential at the right will be lower than that at the left. If on the other hand the positive ions have the greater velocity, then the potential at the right will be positive as compared with that on the left. This latter is what was found to be the case. The potential on the side toward which the arc was deflected was greater than that from which it was deflected. We are, therefore, led to believe that in the arc the positive ions move more rapidly. No. 2.] THE ARC IN A MAGNETIC FIELD. I I I That this should be so in a place as hot as the electric arc is not what one would have expected. Yet reasons for believing that this is possible have been given elsewhere.1 When the anode is very hot we may expect ebullition of the anode with subsequent condensation on the negative ions, causing them to move more slowly. This slow movement would react on the drop in potential at the anode, making it greater. On the other hand where there are negative ions formed with greater ease, as with NaCl, there would be less ebullition and less condensation on the negative ions. This would account for the smaller drop at the anode, for a greater negative discharge to a surrounding cylinder, and for the disappearance of the magnetic effect if, as would appear, this effect is due to the greater velocity of the positive ions. It has also been found that there is less condensation on the negative ions when in a vacuum and that there is a corresponding increase in their velocity.2 This again would explain the smaller drop in potential at the anode, the greater discharge from the arc, and the disappearance of the effect which we have been studying. But on the other hand it would not explain why the effect does not appear in a vacuum in the opposite direction. Therefore, we can not as yet consider this as a complete explanation. Summary. — If two carbon pencils are placed on the opposite sides of an arc, the potential difference between them is changed when the arc is deflected toward one of them by means of a magnetic field. This change may be as high as four volts. The effect can not be produced by blowing the arc to one side, nor by moving the carbon pencils to one side of the arc. Rapidly reversing the field and with each reversal reversing the connection to the voltmeter does not diminish the effect. The effect decreases as the strength of the field is diminished. Changes in the current through the arc do not appreciably influence it. It is, however, increased by lengthening the arc. The effect is smaller with cored than with solid carbons. It decreases and in some cases entirely disappears when salts are » PHYS. R E V . , 15, 346. 2PHYS. REV., 15, 349. C. D. I 12 CHILD. [VOL. XX. placed in the arc, and it could not be detected with an arc between metals. An attempt was made to observe it with graphite terminals, but it was impossible to maintain an arc between such terminals when in a magnetic field. The effect decreases as the pressure on the surrounding gas is diminished and with a pressure of approximately I cm. of mercury it disappears. The common equation for the Hall effect does not apply in this case, since there is here a motion of the ions transverse to the direction of the impressed E.M.F. It is possible, however, to explain almost all of the observations by assuming that the positive ions move more rapidly than the negative ones. COLGATE UNIVERSITY, December, 1904. 

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University of Tasmania Open Access Repository Cover sheet Title Does variation in garden characteristics influence the conservation of birds in suburbia? Author Daniels, G, James Kirkpatrick Bibliographic citation Daniels, G; Kirkpatrick, James (2006). Does variation in garden characteristics influence the conservation of birds in suburbia?. University Of Tasmania. Journal contribution. https://figshare.utas.edu.au/articles/journal_contribution/Does_variation_in_garden_characteristics_influence_the_con Is published in: 10.1016/j.biocon.2006.06.011 Copyright information This version of work is made accessible in the repository with the permission of the copyright holder/s under the following, Licence. If you believe that this work infringes copyright, please email details to: oa.repository@utas.edu.au Downloaded from University of Tasmania Open Access Repository Please do not remove this coversheet as it contains citation and copyright information. University of Tasmania Open Access Repository Library and Cultural Collections University of Tasmania Private Bag 3 Hobart, TAS 7005 Australia E oa.repository@utas.edu.au CRICOS Provider Code 00586B | ABN 30 764 374 782 utas.edu.au 

W2099798365.txt

Current Neuropharmacology, 2007, 5, 180-186 180 Allosteric Modulation of the Calcium-Sensing Receptor Anders A. Jensen and Hans Bräuner-Osborne* Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark Abstract: The calcium (Ca2+)-sensing receptor (CaR) belongs to family C of the G-protein coupled receptors (GPCRs). The receptor is activated by physiological levels of Ca2+ (and Mg2+) and positively modulated by a range of proteinogenic L-
-amino acids. Recently, several synthetic allosteric modulators of the receptor have been developed, which either act as positive modulators (termed calcimimetics) or negative modulators (termed calcilytics). These ligands do not activate the wild-type receptor directly, but rather shift the concentration-response curves of Ca2+ to the left or right, respectively. Like other family C GPCRs, the CaR contains a large amino-terminal domain and a 7-transmembrane domain. Whereas the endogenous ligands for the receptor, Ca2+, Mg2+ and the L-
-amino acids, bind to the amino-terminal domain, most if not all of the synthetic modulators published so far bind to the 7-transmembrane domain. The most prominent physiological function of the CaR is to maintain the extracellular Ca2+ level in a very tight range via control of secretion of parathyroid hormone (PTH). Influence on e.g. secretion of calcitonin from thyroid C-cells and direct action on the tubule of the kidney also contribute to the control of the extracellular Ca2+ level. This control over PTH and Ca2+ levels is partially lost in patients suffering from primary and secondary hyperparathyroidism. The perspectives in CaR as a therapeutic target have been underlined by the recent approval of the calcimimetic cinacalcet for the treatment of certain forms of primary and secondary hyperparathyroidism. Cinacalcet is the first clinically administered allosteric modulator acting on a GPCR, and thus the compound constitutes an important proof-ofconcept for future development of allosteric modulators on other GPCR drug targets. Key Words: Calcium-sensing receptor, CaR, allosteric modulator, calcimimetic, calcilytic, cinacalcet, hyperparathyroidism. INTRODUCTION Calcium (Ca2+) is an essential intracellular messenger and a mediator of a wide range of important physiological and pathophysiological cellular processes. The intracellular concentration of Ca2+ ([Ca2+]i) is at a tightly controlled equilibrium between the actions of various voltage- and ligand-gated calcium ion channels and the release of Ca2+ from intracellular stores, for example as a result of the signalling of certain G-protein coupled receptors (GPCRs). Within the last two decades it has been realized that several cell types also are able to ‘sense’ fluctuations in the extracellular Ca2+ concentration ([Ca2+]o) and that even minute changes in [Ca2+]o elicits an array of cellular responses, such as an increase in [Ca2+]i and the release of various hormones [7, 10, 34, 76]. The cloning of the calcium-sensing receptor (CaR), a GPCR mediating the intracellular responses to extracellular stimuli in the form of Ca2+ (and Mg2+), in 1993 unequivocally established Ca2+ as a first messenger as well as a second messenger [9]. In the subsequent years CaR has been shown to be a unique regulator of metabolic processes in bone, kidney, parathyroid and many other tissues [7, 10, 34, 76]. Thus, the receptor is an attractive target for the treatment of several disorders, and the therapeutic prospects in CaR ligands have been underlined by the recent introduction of cinacalcet (Sensipar and Mimpara in USA and Europe, respectively) in the clinical treatment of uremic secondary hypercalcemia parathyroidism and parathyroid cancer [10, 76]. In recent years the perception of CaR as a metabolic sensor has been substantiated and further expanded by the realization that CaR signalling is potentiated by several L-
-amino acids and by the recent cloning of a related GPCR, GPRC6A, which also mediates the signalling of L-
-amino acids and divalent cations [13, 17, 45, 61, 78]. The physiological importance of Ca2+ sensing and the role of CaR in health and disease have been outlined in several excellent reviews, to which the reader is referred [10, 34, 76]. Following a brief introduction to the structure and signal transduction of CaR, the present review will primarily focus on the pharmacology of endogenous and synthetic allosteric modulators of CaR and their binding modes at the receptor. Finally, the physiological roles played by CaR, and the therapeutic prospects for cinacalcet, the *Address correspondence to this author at Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark; Tel: (+45) 3533 6518; Fax: (+45) 3533 6040; E-mail: hbo@molpharm.net 1570-159X/07 $50.00+.00 first and only allosteric modulator of a GPCR to ever enter the drug market will be discussed. MOLECULAR STRUCTURE AND SIGNAL TRANSDUCTION OF THE CAR The CaR belongs to family C of the GPCR superfamily, a small subfamily comprised by receptors mediating the intracellular metabolic responses to extracellular stimuli in the form of nutrients such as amino acids, ions and taste molecules. Besides CaR family C is constituted by eight metabotropic glutamate receptors (mGluRs), two -aminobutyric acidB receptors (GABABRs), several taste receptors and the recently cloned promiscuous L-
-amino acid receptor GPRC6A [3, 10, 19, 34, 37, 45, 51, 52, 76, 78, 84]. The family C GPCR does not exhibit significant amino acid sequence homology with the other GPCRs, and it shares very few fingerprint residues with other receptors in the superfamily. However, the family C GPCR has the same overall topology as all other GPCRs, as it is constituted by an extracellular amino-terminal domain (ATD), seven transmembrane
-helices (TM1-TM7) connected by intraand extracellular loops (this region will be referred to as ‘the 7TM’), and an intracellular carboxy-terminal (Fig. 1A). Finally, a small region containing nine highly conserved cysteine residues, termed the cysteine-rich region (CRR), is connecting the ATD and the 7TM of CaR. The family C GPCRs exist as constitutive dimeric (or oligomeric) receptor complexes in the cell membrane (Fig. 1A). Whereas the GABAB and taste receptors exist as heterodimeric receptors composed of two different subunits, CaR and the mGluRs form homodimeric complexes via several covalent and noncovalent interactions between the two subunits [2, 3, 47, 52, 57, 67, 72, 73, 77, 84]. The most remarkable structural feature of the family C GPCR is its extraordinarily large extracellular ATD, which consists of ~600 amino acid residues and contains the orthosteric site of the receptor, i.e. the binding site of the endogenous agonist [6, 31]. The crystal structure of the ATD of the mGluR subtype 1 has brought considerable insight into the structure of this region [40, 47, 77]. The ATD consists of two globular lobes arranged in a clam shelf structure, and thus the domain is often referred to as a ‘Venus Flytrap Domain’. The orthosteric site is situated in the cleft between these two lobes, where the agonist binds via interaction to residues located on both sides of this cleft. The location of the orthosteric site in the family C GPCR contrasts that of the rhodopsin-like family A GPCR, where the endoge©2007 Bentham Science Publishers Ltd. Allosteric Modulation of the Calcium-Sensing Receptor Current Neuropharmacology, 2007, Vol. 5, No. 3 181 Fig. (1). A. The homodimeric CaR complex. The ATD, CRR and 7TM regions of the receptor are indicated. The residues in the ATD involved in Ca2+ and L
-amino acid binding and the residues in the 7TM involved in the binding of calcimimetics and calcilytics are given. B. The signal transduction through the CaR homodimer when exposed to Ca2+ alone, Ca2+ and L-
-amino acids, Ca2+ and calcilytics, and Ca2+ and calcimimetics. Inactive and active conformations of the CaR homodimer is given in red and green, respectively. nous agonist binds to a pocket situated within the 7TM of the receptor. Furthermore, G-protein coupling to the family C receptor also occurs to different intracellular receptor regions than to the family A GPCR [62]. Thus, the molecular events underlying signal transduction through the family C GPCR appear to be quite different from those involved in the signalling of other GPCRs, and the fact that the receptors are dimeric complexes seems to be of key importance for their signal transduction [62]. Agonist binding to each of the clefts in the two ATDs of the homodimeric family C GPCR causes the two regions to close up, which in turn elicits a conformational twist in the entire receptor complex believed to rearrange the composition of two 7TM regions and hereby enabling G-protein coupling to the receptor complex (Fig. 1B) [38, 62]. The Orthosteric Site(s) in CaR In the crystal structure of the mGluR1 ATD, L-glutamate binds to the cleft formed by the two lobes through interactions with 13 residues distributed on both sides of the cleft [47, 77]. The endogenous agonists for CaR, Ca2+ and Mg2+, have also been demonstrated to bind to the ATD of the receptor [6, 31], and mutations of several of the residues in CaR corresponding to mGluR1 residues involved in agonist binding have been shown to impact Ca2+- and Mg2+induced signalling through the receptor dramatically [6, 83]. Based on these mutagenesis studies and a homology model of the CaR ATD based on the mGluR1 ATD crystal structure, Ruat and colleagues have recently proposed that Ca2+ binds to CaR by coordination to the polar residues Ser170, Asp190, Gln193, Ser296 and Glu297 with minor contributions from residues Ser147, Tyr218 and Phe270 (Fig. 1A) [74]. The signal transduction through CaR is characterized by a remarkable high cooperativity with Hill coefficients of 3-4 and 2-3 for Ca2+ and Mg2+, respectively [6, 31]. This and the smaller sizes of Ca2+ and Mg2+ ions compared to glutamate, GABA and other agonists for family C GPCRs originally prompted speculations that the orthosteric site in the ATD has to be occupied by more than one Ca2+ or Mg2+ ion in order for CaR to become activated. However, subsequently a small segment in the carboxy terminal of CaR has been proposed to control receptor densitization and influence the cooperativity [24]. Furthermore, in addition to its binding site in the ATD Ca2+ has recently been proposed to act as an agonist at a site situated in the 7TM of the receptor [68]. The presence of orthosteric sites in both the ATD and the 7TM of the receptor would certainly explain its high cooperativity. ALLOSTERIC MODULATION OF CaR Endogenous Allosteric Modulators of CaR L-
-amino acids. The CaR appears to be highly susceptible to allosteric modulation by a wide range of endogenous ligands and environmental conditions. For example, Na+ ion concentration and ion strength [65] as well as changes in environmental pH [64] have been shown to influence CaR signalling significantly. More importantly, however, CaR signalling has recently been shown to be potentiated by numerous L-
-amino acids, in particular the aromatic amino acids L-phenylalanine, L-tyrosine, L-histidine and Ltryptophan [17]. The L-
-amino acids potentiate CaR signalling at physiologically relevant concentrations, and they have been shown to inhibit PTH secretion from human parathyroid cells in an acute and reversible manner [16]. The dual activity of amino acids and 182 Current Neuropharmacology, 2007, Vol. 5, No. 3 Ca2+ at CaR appears to be a feature shared by many family C GPCRs [15]. Several mGluRs and the GABAB receptors have been shown to possess Ca2+ sensing properties [46, 63, 80], and the signalling of the promiscuous L -
-amino acid receptor GPRC6A is also potentiated by Ca2+ and Mg2+ [13, 61]. Interestingly, the L-
amino acids have been demonstrated to exert their effects on CaR signalling through binding to an allosteric site situated in the close vicinity of the orthosteric site in the ATD of the receptor, in fact some receptor residues are involved in both Ca2+ and the L-
-amino acid binding (Fig. 1A) [49, 50, 83]. A triple alanine mutation of the adjacent serine residues Ser169, Ser170 and Ser171, of which Ser170 has been shown to be crucial for Ca2+ signalling through CaR, eliminates the potentiation of receptor signalling exerted by L-
amino acids [83], whereas introduction of the double mutation T145A/S170T in CaR eliminates the ‘L-
-amino acid sensing’ ability of CaR while Ca2+ displays unaltered potency at the mutant compared to the wild type receptor [49]. The apparent concomitant binding of Ca2+ and the L-
-amino acid to two distinct sites both situated in the cleft between the two lobes of the ATD in CaR is in good agreement with studies of mGlu and GABAB receptors, where Ca2+ binding has been shown to take place to neighbouring residues to those involved in L-glutamate and GABA binding, respectively [46, 63]. Furthermore, the realization that CaR is a receptor for both amino acids and divalent cations may also explain why the binding cavity in the ATD of CaR, according to molecular modelling, is of a similar size as those in the mGlu and GABAB receptors [15]. The L-
-amino acids were originally reported to be true allosteric potentiators of CaR in the sense that they were found to be unable to activate CaR in the absence of Ca2+ or other CaR agonists [17]. Subsequently, however, another group has claimed that L– phenylalanine is a CaR agonist capable of eliciting signalling in the absence of calcium [71]. Interestingly, Ca2+ and the L-
-amino acids appear to elicit different intracellular Ca2+ oscillations through CaR, indicating that the receptor response may differentiate depending on the nature of the external stimuli [71, 82]. Ca2+mediated CaR signalling gives rise to high-frequency sinusoidal oscillations upon a raised plateau of [Ca2+]I through a phospholipase C/inositol trisphosphate pathway, and the frequency of these oscillations are negatively regulated by phosphorylation of the intracellular Thr888 residue in the carboxy terminal of CaR by protein kinase C [71, 82]. In contrast, stimulation of the receptor with L -
amino acids, on the other hand, induces transient oscillations characterized by repetitive, low frequency [Ca2+]i spikes that return to the baseline level [71, 82]. These spikes are not induced via phospholipase C/inositol trisphosphate pathway but via the transient receptor potential channel TRPC1 via a signalling cascade involving G
12 proteins, the GTPase Rho, filamin-A and the actin cytoskeleton [70, 71]. In order to explain the different intracellular responses caused by Ca2+ and L-
-amino acids, the two types of agonists have been proposed to stabilize different active conformations of CaR [71]. Considering the close proximity of the binding sites for Ca2+ and the L -
-amino acids in the ATD and the considerable distance from these binding sites to the intracellular Gprotein coupling regions of CaR, it is quite remarkable that the two agonists elicit distinct signalling cascades. Synthetic Allosteric Modulators of the CaR Besides its endogenous agonists Ca2+ and Mg2+, CaR is also activated by other inorganic ions like Gd3+ and Ba2+, polyamines like spermine and spermidine and by the antibiotic neomycin [6, 31, 66]. Explorations into the therapeutic prospects of CaR have been complicated by the chemical nature of these agonists, since they are not obvious lead candidates for medicinal chemistry development. However, this obstacle has been circumvented by the development of several organic compounds modulating CaR signalling via binding to allosteric sites in the receptor. Based on their pharmacological properties on CaR signalling, these synthesised allosteric modu- Jensen and Bräuner-Osborne lators are divided into two classes: calcimimetics and calcilytics. Calcimimetics are allosteric potentiators (or positive allosteric modulators) of CaR, the name referring to their ability to mimic the actions of calcium, whereas calcilytics are allosteric inhibitors (or negative allosteric modulators) of the receptor. Calcimimetics The phenylalkylamine structure fendiline is a relatively weak allosteric potentiator of CaR, which has been used as the lead for the development of a series of more potent calcimimetics, including the compounds NPS R-467 and NPS R-568 (Fig. 2) [56]. NPS R568 has been shown to decrease PTH secretion and increase calcitonin secretion through its activity at CaR on parathyroid cells and C cells, and these effects leads to hypocalcemia due to a reduced efflux of Ca2+ from bone [22, 23, 56]. NPS R-568 and NPS R-467 have been further developed into cinacalcet, which recently has entered the clinic for the treatment of uremic secondary hypercalcemia parathyroidism and parathyroid cancer (see below) [55]. In another series of calcimimetics, medicinal chemistry optimization of N-arylsulfon-1,2-diamine and N-aryl-1,2-diamine structures have resulted in the development of calindol [(R)-2-[N-(1-(1-naphtyl) ethyl)aminomethyl]indole] [20, 42]. As can be seen from (Fig. 2), the structures of the NPS calcimimetics, cinacalcet and calindol are very similar. Calcilytics In spite of some structural similarity to NPS R-568, the calcilytic NPS 2143 (N-[(R)-2-hydroxy-3-(2-cyano-3-chlorophenoxy) propyl]-1,1-dimethyl-2-(2-naphtyl)ethylamine) was developed based on a hit identified in a high throughput screening (Fig. 2) [54]. Being the first published CaR inhibitor, the compound has become an important pharmacological tool to study the physiological functions governed by the receptors [54]. In agreement with the observed effects of calcimimetics, NPS 2143 has been shown to increase PTH secretion from parathyroid cells and to increase plasma levels of Ca2+. A considerable amount of structure-activity work has been performed using NPS 2143 as template. However, variations in the 1,1-dimethyl-2-naphtalen-2-yl-ethylamine part of the molecule have not resulted in more potent calcilytics [26, 81]. Another calcilytic is Calhex 231 (N1-(4-Chlorobenzoyl)-N2 -[1-(1-naphtyl)ethyl]trans-1,2-diaminocyclohexane), one of the most potent CaR inhibitors published to date [41]. Finally, a series of structurally distinct calcilytic compounds not based on the phenylalkylamine-structure have recently been published [1, 35]. Allosteric Sites in the 7TM of CaR The 7TM of the family C GPCR seems to be an attractive target for allosteric modulators, since a plethora of allosteric modulators of mGlu and GABAB receptors have been demonstrated to target this region [21, 25, 39]. Analogously, many of the published synthetic allosteric modulators of the CaR have been shown to target the 7TM of the receptor [30, 32, 36, 48, 59, 60], and the residues involved in the binding of the calcilytics NPS 2143 and Calhex 231 and the calcimimetics NPS R-568 and calindol have been outlined in further detail in mutagenesis studies based on homology models of the 7TM of CaR built using the crystal structure of the family A GPCR rhodopsin as template (Fig. 1A) [48, 59, 60]. NPS 2143 and Calhex 231 both bind to the extracellular part of the 7TM of CaR, and their binding sites are largely overlapping although minor differences in the binding modes of the two calcilytics exist. The binding of both ligands is anchored in a salt bridge from the protonated secondary amino group in the ligands to the Glu837 residue in TM7 [36, 48, 60]. In addition to this interaction, the aromatic ring systems in NPS 2143 is believed to form hydrophobic contacts and stacking with the TM2 residue Phe668 and the TM3 residues Phe684 and Phe688, and the Arg680 residue in TM3 has been proposed to interact with the hydroxy group of the ligand [48, 59]. Finally, mu- Allosteric Modulation of the Calcium-Sensing Receptor H N Current Neuropharmacology, 2007, Vol. 5, No. 3 H N F3C O 183 H N N H R NPS R-467 (R = H) NPS R-568 (R = Cl) Cinacalcet Cl Calindol CN N H Cl NH OH H N O O Calhex 231 NPS 2143 O O O O O O HN O O N HN N H N OH O O NH2 O BMS compound 1 JKJ05 Fig. (2). Chemical structures of synthetic allosteric modulators of CaR. tation of Ile841 located one
-helix turn below Glu837 in TM7 of CaR has also been found to result in reduced inhibitory potency of NPS 2143 [59]. Phe684, Phe688 and Ile841 are also important for Calhex 231 binding to CaR but the inhibitory effects of this calcilytic is not affected by an alanine mutation of Arg680, and furthermore it is less affected by a F688A mutation in CaR than NPS 2143. On the other hand, the antagonistic potency of Calhex 231 is reduced by an alanine mutation of Trp818 in TM6 of CaR, whereas that of NPS 2143 is not [59]. Finally, alanine substitutions of Leu776 and Phe821 in TM5 and TM6 of CaR, respectively, result in increased antagonistic potency of Calhex 231, whereas NPS 2143 does not display changes in its IC50 values at these mutants compared to the wild type receptor [59]. has reported that binding of the compound to CaR does not involve the Glu837 residue in TM7 shown to be crucial for the binding of all of the phenylalkylamine-based allosteric modulators, whereas mutation of the Ile841 residue eliminates the inhibitory effects of BMS compound 1 on CaR signalling [35]. Hence, this calcilytic appear to bind to an allosteric site in the 7TM of CaR, which is in close proximity to but does not overlap with the site for the phenylalkylaminebased modulators. Furthermore, the fact that the allosteric inhibitor JKJ05, a closely related analogue of BMS compound 1, was converted into an allosteric potentiator with the introduction of the E837A mutation into CaR, further illustrates the subtle molecular differences in the 7TM conformations stabilized by calcilytics and calcimimetics [35]. The binding of the two calcimimetics NPS R-568 and calindol to CaR are also centered around an ionic interaction between the charged nitrogens of the compounds and Glu837 in TM7 [59]. However, in contrast to their involvement in calcilytic binding, the TM3 residues Arg680, Phe684 and Phe688 are not important for the binding of the calcimimetics. Instead, TM6 has been proposed of key importance for their ability to potentiate CaR signalling, as the potentiating effects on CaR signalling by NPS R-568 and calindol are eliminated by mutations of the Trp818 residue and the Phe821 residue, respectively [59]. It is interesting that the four transmembrane helices involved in agonist binding to the family A GPCR, TM3, TM5, TM6 and TM7, also form the binding pocket for the allosteric modulators in CaR and other family C GPCRs. This raises the question whether the allosteric potentiators in fact are allosteric agonists with intrinsic activities at CaR in the absence of Ca2+ and Mg2+, as it has been reported to be the case for allosteric potentiators of mGlu and GABAB receptors [4, 28]. In a recent study, calindol has been demonstrated to be an agonist at a CaR construct, where the ATD and the carboxy terminal of the receptor have been truncated [69]. Although no calcimimetics have displayed intrinsic activity at the wild type receptor the agonism displayed by calindol at this artificial CaR construct underlines the similarities between the orthosteric site in the rhodopsin-like family A GPCR and the common allosteric site for the phenylalkylamine-based calcimimetics and calcilytics in CaR. Whereas all published allosteric modulators of CaR appear to bind to the 7TM of the receptor, synthetic allosteric modulators targeted to the ATD of the receptor could also be envisioned. As mentioned below, a considerable number of somatic Considering that NPS R-568, NPS 2143, Calindol and Calhex 231 are structurally related phenylalkylamines all having a positively charged amino group, it is not surprising that they target a common allosteric site in the 7TM of CaR. Interestingly, a structurally distinct calcilytic from Bristol-Meyers Squibb (‘BMS compound 1’ in Fig. 2) has recently been shown not to compete with the binding of a tritiated NPS 2143 analogue to CaR, indicating that this compound has a different site of action than NPS 2143 and the other phenylalkylamines [1]. In support of this, the Spiegel group 184 Current Neuropharmacology, 2007, Vol. 5, No. 3 mutations in the CaR gene linked to various disorders have been shown to alter the signalling properties of the receptor, and this indicates that the entire receptor protein is susceptible to allosteric modulation. Furthermore, the allosteric potentiation of CaR signalling by L-
-amino acid binding to a site close to the orthosteric site in the ATD further supports this notion. PHYSIOLOGICAL ROLES OF CAR Due to the significance of calcium as a primary and secondary messenger, the extracellular calcium level in the blood is very tightly controlled via regulation of dietary uptake, renal excretion and bone metabolism. Thus an elevated calcium level will lead to decreased renal calcium resorption, decreased intestinal absorption, increased bone formation and decreased bone resorption, which at least to some extend is caused either directly or indirectly (via parathyroid hormone (PTH) and calcitonin hormones) by activation of CaR [8]. In accordance with the broad expression of CaR, the receptor might also possess other important physiological roles such as regulation of gut hormone secretion [14] and control of arterial blood pressure [79], but these roles remains to be studied in details. In the parathyroid glands, CaR has been identified as a central regulator of the synthesis and secretion of PTH and of parathyroid cellular proliferation. Thus, a decrease in serum calcium level will lead to increased secretion of PTH, which in turn will act on (1) the kidney to promote calcium reabsorption and synthesis of 1,25(OH)2D3 (a derivative of vitamin D3 which promotes intestinal absorption of calcium and phosphate) and (2) bone to release skeletal calcium. These effects are further augmented by concomitant decrease in secretion of calcitonin from thyroid C-cells, which has the opposite effect of PTH on bone metabolism and kidney reabsorption [8]. CaR is also expressed in the kidney, bone and intestine and might thus also contribute directly to the physiological effects outlined above in addition to the indirect effects via PTH and calcitonin, but the significance of these direct effects are still being debated intensely. The physiological importance of CaR on calcium homeostasis and PTH secretion has been elucidated by analysis of genetically modified mice and analysis of humans with inherited or acquired disorders involving the CaR. More recently, the use of calcimimetics and calcilytics in vivo has also contributed significantly to our knowledge (see next section). CaR knock-out mice display highly elevated PTH levels, despite significantly elevated serum calcium levels, and parathyroid hyperplasia [33], which all point to the direct effects of CaR on the control of PTH secretion and parathyroid cellular proliferation. A second line of evidence comes from identification of numerous mutations in the CaR gene identified from individuals suffering from three different genetic disorders, Familial Hypocalciuric Hypercalcemia (FHH), Neonatal Severe Hyperparathyroidism (NSHPT) and Autosomal Dominant Hypocalcemia (ADH). Pharmacological characterization of these somatic mutations in CaR mutants heterogeneously expressed in mammalian cell lines have shown that introduction of the FHH and NSHPT mutations in CaR result in reduced potencies of Ca2+ at the receptor, whereas ADH mutants display increased Ca2+ potencies. Collectively, the studies provide a compelling explanation for the clinically observed elevated and decreased serum calcium levels in FHH/NSHPT and ADH, respectively [12]. A third line of in vivo evidence comes from autoimmunity disorders in which patients have developed anti-CaR antibodies that either inhibit [44] or activate [43] CaR and thereby produce clinical manifestations resembling FHH or ADH, respectively. Finally, patients with either primary or uremic secondary hyperparathyroidism (PHPT and SHPT, respectively) show significant reduced expression of CaR in the parathyroid gland [18, 27], which at least in part provide a possible explanation for the observed increase in PTH secretion. Jensen and Bräuner-Osborne THERAPEUTIC PROSPECTS Recently, North American and European drug regulatory authorities approved the calcimimetic cinacalcet for the treatment of SHPT in patients with end-stage renal disease receiving hemodialysis and of PHPT caused by parathyroid cancer. Both diseases are characterized by elevated PTH and calcium levels as well as other disturbances in mineral metabolism, which leads to bone diseases and other complications [11, 75]. Several clinical trials have been conducted with cinacalcet and other calcimimetics, which have shown that the compounds are very effective in reducing the elevated PTH and calcium levels in various forms of PHPT and SHPT [5, 8, 58]. So far the drug has only been approved for the previously mentioned forms of PHPT and SHPT, but it is predicted that the drug will also be approved for other forms of hyperparathyroidism in the future [8]. In addition the drug might also be approved for the treatment of FHH, NSPHP and hypercalcemia caused by autoantibodies that inhibit the CaR [8]. Only few pre-clinical studies have been conducted with calcilytics and none have yet been approved for human use. Two studies with NPS 2143 have shown that the compound leads to a 3-4 fold increase in PTH levels lasting for several hours causing an increase in bone turnover, but not an increase in bone mineral density (BMD) [29, 53]. Given that injections of PTH is approved for the treatment of osteoporosis, it has been suggested that short acting calcilytics, causing bursts of PTH levels similar to the injected PTH rather than the sustained increases seen with NPS 2143, could lead to increases in BMD [29, 53]. BMS compound 1 has such a short acting profile in vivo in rats resulting in PTH bursts [1], but the effect of the compound on BMD has yet to be studied and the hypothesis of use of calcilytics for treatment of osteoporosis thus remains to be proven. Other putative indications for calcilytics are treatment of ADH and hypocalcemia caused by autoantibodies that activate the CaR [8]. CONCLUSIONS Since the cloning of the CaR in 1993 a wealth of information on its structure, function, pharmacology and physiology has been generated. The receptor has been shown to be a central regulator of the secretion of PTH and an important feed-back sensor to control serum calcium levels, and this has formed the rationale for the development of cinacalcet for treatment of certain forms of PHPT and SHPT. Cinacalcet is a positive allosteric modulator of CaR and is the first compound with such a pharmacological profile on a GPCR to obtain regulatory approval as a drug. As such, the compound is very important as it has demonstrated proof-of-concept for the many other allosteric GPCR modulators that are currently in drug development. It is to be expected that the indications of cinacalcet will be broadened in the future, and that other calcimimetics will be marketed. Whether a calcilytic will also reach the market to treat e.g. osteoporosis is less certain as additional pre-clinical studies using short-acting analogues are needed to show proof-of-concept. So far, the physiological function of CaR is best known in the parathyroid gland and the kidney. However, the receptor shows a much wider expression pattern and additional physiological functions have yet to be fully investigated. The development of allosteric modulators that can be applied in vitro and in vivo will undoubtedly accelerate such studies as exemplified by a recent study suggesting a role of CaR in the control of arterial blood pressure [79]. In addition the physiological importance of the allosteric modulation of CaR by L-
-amino acids remains to be fully elucidated. In that respect, it would be interesting to further elucidate this allosteric site by characterization of series of analogues of the aromatic L -
-amino acids, which could hopefully lead to development of potent and selective compounds that could be used to study the physiological importance of L-
-amino acid sensing by the CaR. Furthermore, considering the reports demonstrating differen- Allosteric Modulation of the Calcium-Sensing Receptor tial CaR signaling exerted by Ca2+ and L-
-amino acids [68, 69, 80], different calcimimetics could be envisioned to elicit different physiological responses. Finally, albeit higher levels of Ca2+ or Mg2+ are needed to modulate/activate GPRC6A than CaR [13, 45, 61] it is possible that GPCR6A has a physiological role as a divalent cation sensor in e.g. bone where higher concentrations are present than in blood. Further studies using selective ligands and/or genetically modified mice are needed to elucidate the physiological role of this novel receptor. Current Neuropharmacology, 2007, Vol. 5, No. 3 [19] [20] [21] [22] ACKNOWLEDGEMENTS We would like to express our gratitude to the many group members that have contributed to our research on family C GPCRs during the last decade. Our work has been supported by grants from the Danish Medical Research Council (H.B.-O. and A.A.J.), the Novo Nordisk Foundation (H.B.-O.) and the Lundbeck Foundation (A.A.J.). [23] [24] REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] Arey, B.J., Seethala, R., Ma, Z., Fura, A., Morin, J., Swartz, J., Vyas, V., Yang, W., Dickson, J.K., Jr., Feyen, J.H. (2005) A novel calcium-sensing receptor antagonist transiently stimulates parathyroid hormone secretion in vivo. Endocrinology, 146, 2015-2022. Bai, M., Trivedi, S., Brown, E.M. (1998) Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell surface of CaR-transfected HEK293. J. Biol. Chem., 273, 23605-23610. Bettler, B., Kaupmann, K., Mosbacher, J., Gassmann, M. (2004) Molecular structure and physiological functions of GABAB receptors. Physiol. Rev., 84, 835-867. Binet, V., Brajon, C., Le Corre, L., Acher, F., Pin, J.P., Prezeau, L. (2004) The heptahelical domain of GABAB2 is activated directly by CGP7930, a positive allosteric modulator of the GABAB receptor. J. Biol. Chem., 279, 29085-29091. Block, G.A., Martin, K.J., de Francisco, A.L., Turner, S.A., Avram, M.M., Suranyi, M.G., Hercz, G., Cunningham, J., Abu-Alfa, A.K., Messa, P., Coyne, D.W., Locatelli, F., Cohen, R.M., Evenepoel, P., Moe, S.M., Fournier, A., Braun, J., McCary, L.C., Zani, V.J., Olson, K.A., Drueke, T.B., Goodman, W.G. (2004) Cinacalcet for secondary hyperparathyroidism in patients receiving hemodialysis. N. Engl. J. Med., 350, 1516-1525. Bräuner-Osborne, H., Jensen, A.A., Sheppard, P.O., O'Hara, P., KrogsgaardLarsen, P. (1999) The agonist-binding domain of the calcium-sensing receptor is located at the amino-terminal domain. J. Biol. Chem., 274, 1838218386. Brown, E.M. (1991) Extracellular Ca2+ sensing, regulation of parathyroid cell function, and role of Ca2+ and other ions as extracellular (first) messengers. Physiol. Rev., 71, 371-411. Brown, E.M. (2007) Clinical lessons from the calcium-sensing receptor. Nat. Clin. Pract. Endocrinol. Metab., 3, 122-133. Brown, E.M., Gamba, G., Riccardi, D., Lombardi, M., Butters, R., Kifor, O., Sun, A., Hediger, M.A., Lytton, J., Hebert, S.C. (1993) Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid. Nature, 366, 575-580. Brown, E.M., MacLeod, R.J. (2001) Extracellular calcium sensing and extracellular calcium signaling. Physiol. Rev., 81, 239-297. Chan, A.K., Duh, Q.Y., Katz, M.H., Siperstein, A.E., Clark, O.H. (1995) Clinical manifestations of primary hyperparathyroidism before and after parathyroidectomy. A case-control study. Ann. Surg., 222, 402-412. Chattopadhyay, N., Brown, E.M. (2006) Role of calcium-sensing receptor in mineral ion metabolism and inherited disorders of calcium-sensing. Mol. Genet. Metab., 89, 189-202. Christiansen, B., Hansen, K.B., Wellendorph, P., Bräuner-Osborne, H. (2007) Pharmacological characterization of mouse GPRC6A, an L-
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sustainability Article Elderly Suitability of Park Recreational Space Layout Based on Visual Landscape Evaluation Weiyi Yu 1, *, Hong Hu 1 and Bindong Sun 2,3, * 1 2 3 *



 Citation: Yu, W.; Hu, H.; Sun, B. Elderly Suitability of Park Recreational Space Layout Based on Visual Landscape Evaluation. Sustainability 2021, 13, 6443. https:// doi.org/10.3390/su13116443 Academic Editors: Abdul Raouf and Tayyab Shah School of Mathematics and Computer Science, Shangrao Normal University, Shangrao 334001, China; 305138@sru.edu.cn Research Center for China Administrative Division, East China Normal University, Shanghai 200241, China School of Urban and Regional Science, East China Normal University, Shanghai 200241, China Correspondence: 314115@sru.edu.cn (W.Y.); bdsun@re.ecnu.edu.cn (B.S.) Abstract: Urban parks are critical for sustainable urban development. They are of theoretical and practical significance for analyzing the visual landscape of recreational spaces from the perspective of the elderly. This analysis can be used for exploring new methods for optimizing recreational space layouts to improve the physical and mental health of the elderly in parks, thus realizing the sustainable development of urban society. Taking Ziyang Park in Shangrao Central District of Jiangxi Province, China, as an example, starting from the visual characteristics of the elderly, this study quantitatively calculated the landscape viewability, total view ratio, and water view ratio for the elderly in each recreational space using Python Scripting for ArcGIS. We briefly express the elderly suitability of the visual landscape for each recreational space through a weighted synthesis of the calculation results. Our findings show that, in Ziyang Park, the elderly suitability of the visual landscape for recreational spaces is not only low overall, but also gradually decreases from the interior to the exterior of this park. Moreover, this spatial distribution may be caused by the location of zoning, surface elevation, and road slopes, as well as the individual characteristics of each recreational space. Finally, we discuss the requirements of the elderly for some geographical factors, along with the feasibility of using ArcGIS 3-D analysis to optimize the layout of the park recreational space, with the aim of providing a new research perspective and an effective reference method for designing layouts of such spaces that are favorable for the elderly to better guarantee the sustainable development of urban society. Keywords: visual landscape; park; recreational space; elderly suitability; GIS; Python; sustainable development Received: 2 May 2021 Accepted: 3 June 2021 Published: 5 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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/). 1. Introduction The world’s population is aging [1]. By 2030, the proportion of people over 60 in the United States, Japan, and the United Kingdom will increase to 26.1, 27.8, and 37.3%, respectively [2]. In China, due to the large population base, longer life expectancy, and continuous decrease in the population of child-bearing age, the proportion of the elderly population may rise to 25% or higher [3]. Parks are the most commonly visited public green spaces for the elderly in the city. They play an indispensable role in maintaining the physical and mental health of the elderly and promoting social interaction. Therefore, faced with the severe trend in population aging, an increasing number of scholars have begun to focus on the elderly suitability of parks (also referred to as “aging-suitability” [4], which essentially reflects the suitability of parks for the elderly) and have aimed to improve this suitability to ameliorate the health and living conditions of the elderly, finally promoting the sustainable development of urban society [4,5]. However, a large number of studies have shown that 80% of people’s perception of spatial information occurs through their vision [6–8], meaning that the visual landscape of spaces can greatly affect our judgment of its suitability. Visually attractive outdoor Sustainability 2021, 13, 6443. https://doi.org/10.3390/su13116443 https://www.mdpi.com/journal/sustainability Sustainability 2021, 13, 6443 2 of 17 spaces can often result in a higher or better perceived suitability [9]. However, at present, scholars have considered the suitability of parks for the elderly only from the perspective of the facility configuration [5,10–12], space security [5,12], space accessibility [5,13], space sociality, environmental comfort [5,14], and space diversity and scale [15]. This shows that although there is evidence that the elderly have high-quality visual needs for park space [14], there is no research to date that evaluates the elderly suitability of parks from the perspective of vision or visual landscapes. In recent decades, as the visual landscape of a space will affect, to a significant extent, people’s judgment of the suitability of that space, some scholars and organizations (such as the American Society of Landscape Architects) have attempted to improve outdoor space suitability, as well as examining the theme of space visual landscape and identifying many visual landscape evaluation methods. These methods mainly include field observation scoring [16], network questionnaire surveys [17–19], computer image analysis [20], expert evaluation [19,21], the analytic hierarchy process and scenic beauty evaluation method [22], and geographic information systems (GIS) comprehensive analysis [23]. However, as noted from existing visual landscape research, there is also no specific precedent to optimize the elderly suitability of parks based on a visual landscape evaluation and analysis. To summarize, current research on the suitability of parks for the elderly, whether for evaluation or optimization, lacks thought and practice from the vision or visual landscape dimension. However, the visual perception ability of the elderly (such as visual distance and visual acuity, among others) is generally not as good as that of the young [8]. Therefore, in a park, if the layout, quantity, and terrain of its spaces are not reasonable, the elderly may suffer more visual interference or obstacles than the young in the same space. Accordingly, their visual landscape (such as the landscape scope and angle) based on this space will also be limited, inevitably affecting their quality of life, even hindering the sustainable development of urban society [24]. Therefore, referring to the view of Ma [25], i.e., adjusting the spatial relationship of urban landscapes can eliminate or reduce the spatial resistance to urban sustainable development, attention must be focused not only on the degree and characteristics of park landscape beauty, but also on the elderly’s visual perception suitability of the layout, quantity, and terrain of the park spaces [26]. Further, these aspects should be taken as the basis for optimizing the spatial layout of parks and improving the suitability of parks for the elderly. Lorenzo [27] and Zhang [23] show that the most critical technology restricting the evaluation and analysis of the visual landscape is the simulation and analysis of spatial visual relationships; GIS software can just meet these technical requirements. However, this paper holds that the efficiency of previous studies that have used GIS to manually complete visual landscape evaluation and analysis is excessively low. Therefore, to compensate for the above deficiencies, this study takes Ziyang Park in Shangrao Central District of Jiangxi Province, China, as an example. Based on the concept and evaluation index system of the elderly suitability of the visual landscape of park recreational spaces, this study evaluates and analyzes the elderly suitability of the visual landscape of the recreational spaces in Ziyang Park using Python Scripting for ArcGIS; we discuss the feasibility of optimizing the layout of these spaces using ArcGIS 3-D analysis, combined with the requirements of the elderly for some terrain factors. This study aims to provide a new research perspective and an implementation method for designing layouts of park recreational spaces that are favorable for the elderly to better guarantee the sustainable development of urban society. The remainder of the study is organized as follows: Section 2 provides a literature analysis on the elderly suitability of parks and the spatial visual landscape. Section 3 demonstrates the materials and methods used in this study. Section 4 illustrates the results of the evaluation, analysis, and optimization. Finally, Section 5 presents the discussion and conclusions. Sustainability 2021, 13, 6443 3 of 17 2. Related Literature The visual landscape is one of the main topics in the field of landscape science, but its definition remains ambiguous across academic circles. A few scholars use “visual environment of landscape” or “visual environment” to express the “visual landscape”, but all refer to people’s visual perception of the objective environment and tend to focus more on the visual attributes of landscapes [28]. A systematic literature review shows that existing visual landscape research can be divided into three categories: visual landscape quality evaluation, visual landscape preference research, and visual impact evaluation [29]. Based on this categorization, the relevant literature from the past 20 years was reviewed; the following key points summarize our findings: 1. 2. 3. Some scholars engaged in park landscape research have mainly focused on the forest edge landscape and plant landscape of parks. Only a few scholars have conducted research on the visual landscape of park recreational spaces [30] and main pedestrian corridors [31,32], where they only focused on the relationship between the visual preference or visual influence of visitors and the landscape designs or operations of scenic spots. Among the existing visual landscape evaluation methods, the most commonly used is the scenic beauty estimation method (SBE), followed by the expert evaluation method. However, due to a lack of on-site experience, the SBE sometimes produces deviations; the expert evaluation method excessively relies on the subjective judgment of professionals, such that its evaluation results are one sided. In contrast, the more comprehensive GIS analysis method can partially improve upon the objectivity and accuracy of the evaluation [23]. In terms of visual landscape preference research and visual impact evaluation, Cakci [19] stated that the orderliness, spatiality, maintainability, and prominence of landscape elements directly affect the users’ landscape preferences. Yao [9] found that the visual perception of green landscapes in rural residential areas is affected by the vegetation type, vegetation area, color contrast, green space openness, terrain type, and positive human factors. Scarfò [31] evaluated the visual impact of forest operations on the landscape through a GIS analysis of three landscape features (visibility, forest landscape quality, and visual sensitivity) in the main tourism corridors of a reserve. Polat [30] found that the water area, widths of pedestrian passages, and functions of leisure areas have a positive impact on the visual quality of the landscape area. Qi [33] stated that the advantage of country park landscapes lies in their sufficient water landscape construction space, whereas a disadvantage is the visual interference caused by surrounding high-rise buildings. Liu [16] reported that the sight distance, visual disturbance frequency, green looking ratio, inter-visibility, openness, and the relative angle and relative area of the artificial matrix are seven specific indicators that affect the visual landscape quality of country parks. As mentioned in Section 1, there is still a lack of elderly suitability evaluations of parks from the perspective of vision or visual landscape. However, for the optimization aspect of the elderly suitability of parks, Carstens [34] proposed that an elderly community should establish external space and facilities “suitable for the elderly” to adapt to all aspects of the elderly’s abilities. Marcus [35] proposed that a “therapeutic” garden should be set up in a community for the elderly according to the health care needs of the elderly. Cao [36] stated that urban public spaces for the elderly should be designed according to the “5D” strategy (density, diversity, pedestrian space, accessibility, and fault tolerance). Thus, the main starting points for scholars to optimize the elderly suitability of parks are the health needs, behavioral ability, and characteristics of the elderly. Sustainability 2021, 13, 6443 4 of 17 In summary, there is little research on how to scientifically develop and plan park spaces according to the vision or visual landscape; moreover, there is an even greater lack of research on how to evaluate and optimize the elderly suitability of a park based on the visual ability characteristics of the elderly and their visual landscape. From the viewer’s perspective, combined with the spatial distribution of the landscape and its surrounding facilities, the analysis of the visual field characteristics that visitors can see in the process of visiting a park is an important basis for the development and planning of urban parks [26,29]. Therefore, combined with the above literature analysis, this paper takes the most commonly used recreational space for the elderly as a study example. The selected recreational spaces include stone/wood benches, characteristic pavilions, cultural corridors (i.e., corridors designed to convey cultural characteristics), and hydrophilic platforms (i.e., platforms extending from land onto a water surface, accessible for recreation), among others. Such spaces are mostly distributed in the interior of landscape points (some landscape points are also considered recreational space) or on both sides of the main walkways. 3. Materials and Methods 3.1. Research Area Shangrao is located in northeastern Jiangxi Province, China, at the intersection of the Yangtze River Delta Economic Zone, Haixi Economic Zone, and Poyang Lake Ecological Economic Zone. Since ancient times, Shangrao has been known as an “excellent and rich city of ecology” and “thoroughfare in all directions”. In 2016, Shangrao Central District was designated as the provincial sub-central city of Jiangxi Province, with a permanent resident population of approximately 1 million; the elderly population above 60 years accounted for approximately 12.74% of the city’s population. This shows that the degree of population aging in Shangrao Central District is relatively high. Ziyang Park is located in northeastern Shangrao Central District, bordering Jiyang Road to the east, Chasheng Road to the south, Guihua Road to the west, and Fenghuang Avenue to the north. It covers an area of approximately 8.78 hectares, of which the water area accounts for approximately 21.52%; the park is a monument to the memory of Zhu Xi, a famous philosopher and educator of the Southern Song Dynasty. The functional space is composed of five areas: the main entrance square, children’s recreation, water activity, mountain amusement, and culture and history exhibition areas (Figure 1). In terms of the external spatial relationships, Ziyang Park is adjacent to each residential district in all directions other than to the west and north, which are adjacent to the commercial city (Figure 2). Therefore, Ziyang Park is a public outdoor space with a strong living atmosphere in this area. Pavilions (such as the Ziyang Pavilion, Liuhua building, Zhizhi Pavilion, Meihua Pavilion, Zhulu Pavilion, Miaowan teashop, and Pleasure-boat Pavilion, among others), with different styles, are the favorite attractions of the elderly in Ziyang Park. They provide a strong cultural atmosphere, meeting the needs of the elderly to enjoy the calligraphy of famous masters and cherish the memory of the sages. On the winding wooden bridge in the park, the elderly can also observe fish in the lake and interact with nature. Therefore, Ziyang Park is a suitably representative choice as the research case. Sustainability 2021, 13, 6443 Sustainability 2021, 13, x FOR PEER REVIEW Sustainability 2021, 13, x FOR PEER REVIEW 5 of 17 5 of 16 5 of 16 Figure 1. 1. Sketch Sketch of of the the internal internal structure structure of of Ziyang Ziyang Park. Park. Figure Figure 1. Sketch of the internal structure of Ziyang Park. Figure 2. External spatial relationship of Ziyang Park. Figure2.2.External Externalspatial spatialrelationship relationshipof ofZiyang ZiyangPark. Park. Figure 3.2. Research Data 3.2. 3.2.Research ResearchData Data Two types of data were used in this study. First, general attribute data on various Two types data were used ininthis study. general attribute data on Two typesofof were used this study.First, First, general onvarious various ground elements in data the case park, mainly including the name andattribute categorydata of each ground ground elements ininthe case park, mainly including the name and category of each ground ground elements the case park, mainly including the name and category of each element, were obtained from the field investigation. Second, geospatial data onground roads, element, obtained from the Second, geospatial data on element, were were from thefield fieldinvestigation. investigation. Second, geospatial datapark on roads, roads, buildings, lakes,obtained landscapes, pavilions, benches, and other facilities in the case were buildings, lakes, landscapes, pavilions, benches, and other facilities in the case park were buildings, lakes, landscapes, pavilions, benches, and other facilities in the case park were considered. Among them, 2D geospatial data were mainly sourced from the Shangrao considered. Among them, 2D geospatial data were mainly sourced from the Shangrao considered. Among them, 2D geospatial data were mainly sourced from the Shangrao tourism map compiled by the Jiangxi map publishing station in September 2013; it was tourism compiled by Jiangxi map publishing station 2013; ititwas tourismmap map compiledpaper bythe the Jiangxi publishing stationin inSeptember September 2013;ArcGIS was obtained by scanning maps andmap performing classified vectorizing using obtained by scanning paper maps and performing classified vectorizing using ArcGIS obtained by scanning paper maps and performing classified vectorizing using ArcGIS 10.4. The 3-D DEM data were mainly obtained using hand-held global positioning system 10.4. 3-D data were obtained using hand-held global positioning system 10.4.The The 3-DDEM DEMas data weremainly mainly obtained using hand-held global positioning system tools. In addition, addition, mentioned in Section Section 1, the the evaluation should first consider elderly elderly tools. In as mentioned in 1, evaluation should first consider tools. In addition, as mentioned in Section 1, the evaluation should first consider elderly sightline barriers barriersto to simulatethe thegeographical geographicalenvironment environment elderly. Therefore, ussightline ofof thethe elderly. Therefore, using sightline barriers tosimulate simulate the geographical environment of the elderly. Therefore, using the above-mentioned geospatial data, the tin surface of Ziyang Park, which is also the above-mentioned geospatial data, the tinthe surface of Ziyang Park, which iswhich also aispart ofaa ing the above-mentioned geospatial data, tin surface of Ziyang Park, also part of the geospatial data used in this study, was created. the geospatial data used in this created. part of the geospatial data usedstudy, in thiswas study, was created. Sustainability 2021, 13, 6443 6 of 17 3.3. Research Methods 3.3.1. Evaluation Indices and Weights According to the aforementioned literature analysis, there are many factors that affect the visual landscape, but many studies on quality are based on subjective judgment. Therefore, to further understand the needs of the elderly in the park visual landscape and the factors influencing the park visual landscape for the elderly, we designed a paper questionnaire according to the research in the literature [5,8,14,34,37–39] and conducted a questionnaire survey on some elderly people in five local parks. A total of 90 questionnaires were issued, and 74 valid questionnaires were collected. The statistical results show that the visual distance, landscape visibility, landscape aesthetic degree, landscape scale, size of the visual field, and the proportion of the water area in the visual field are the main factors influencing the park visual landscape for the elderly. The broad visual field, high landscape visibility, beautiful landscape, a certain size for the landscape scale, and an appropriate proportion of waterscapes are the main needs of the elderly in the park visual landscape. Based on this, combined with the research methods used in a previous study [40] and after removing some non-spatial factors and the elderly’s own factors, we set three influencing factors for the elderly’s visual landscape in park recreational spaces: landscape viewability, total view ratio, and water view ratio. We then proposed that the landscape viewability for the elderly (ELV) of a recreational space refers to the proportion of the number of landscapes that the elderly can see in this recreational space with respect to the total number of park landscapes. The total view ratio for the elderly (ETVR) of a recreational space refers to the proportion of the total surface area that the elderly can see in this recreational space with respect to the total surface area of the park. The water view ratio for the elderly (EWVR) of a recreational space refers to the proportion of the water area that the elderly can see in this recreational space with respect to the total water area of the park. Second, we defined the weighted synthesis of the landscape viewability, the total view ratio, and the water view ratio for the elderly in a recreational space as the elderly suitability of the visual landscape of this recreation space (ESVL). Finally, based on evaluations by 20 experts and an analysis using the analytic hierarchy process (AHP), we obtained the weight coefficients of the ELV, ETVR, and EWVR in the ESVL evaluation of park recreational spaces, which were 0.36, 0.52, and 0.12, respectively, as follows: ESVL = ELV × 0.35 + ETVR × 0.52 + EWVR × 0.12. (1) 3.3.2. Evaluation Hypotheses To ensure authenticity and availability, three minor hypotheses were applied in the evaluation stage, as follows: 1. 2. 3. The elevation of a recreational point, i.e., the elevation of an observation point, was assigned according to the principle of “the ground elevation of the recreation point + 1.2 m (which is the approximate height of an elderly man’s eyes above the ground when he sits down)”. A landscape point, i.e., a target point, was represented by the centroid point of its ground plane; its elevation was assigned according to the principle of “the centroid point’s elevation + the landscape’s height”. Referring to the field investigation results, the longest distance for the elderly to recognize the type of scenery was assumed to be 170 m. 3.3.3. Measurement of Evaluation Indices by Python Scripting for ArcGIS ArcPy is a Python site package launched by ESRI after ArcGIS 10.0 containing 91 functions, 37 classes, and 5 modules; it allows users to flexibly create simple or complex workflows through Python Scripting for ArcGIS, aiding in the efficient automation of geographic processing tasks [5,41–43]. Therefore, referring to the manual process of using ArcGIS intervisibility analysis and viewshed analysis tools [44], as well as documents on Sustainability 2021, 13, 6443 7 of 17 the use of the ArcPy function, this study measured the above three evaluation indices according to the following methods. (1) Measurement of Landscape Viewability for the Elderly We first obtained the line network of sight from each recreational point to each landscape point by loading the layers of the recreational points, landscape points, and tin surface, and then using the Python script (Appendix A.1) to call the construct sightlines [45] and intervisibility [46] functions of ArcPy. Then, after excluding some lines of sight that were more than 170 m in length, we obtained the landscape viewability for the elderly in any recreational space by classifying and summarizing the attribute data for the sight line network. (2) Measurement of the Total View Ratio and Water View Ratio for the Elderly By converting the park tin surface to the park grid surface, loading the layers of the recreational and landscape points, and using the following two steps in the Python script (Appendix A.2), we obtained the total view ratio for the elderly in any recreational space according to the definition (Section 3.3.1). Step 1: call the select_analysis function of ArcPy to separate the Shp layer with 16 recreational points into 16 single point layers. Step 2: call the viewshed [47] function of ArcPy to solve the grid cells intersecting the park grid surface and each recreational point layer, so as to obtain the total view area (counted by the number of grids) for the elderly in any recreational space. Then, using the Python script (Appendix A.3) to call the extractbymask function of ArcPy, the grid cells intersecting any total view area and the park lake surface were solved to obtain the water viewshed area for the elderly in any recreational space. Finally, according to the definition given in Section 3.3.1, we further obtained the water view ratio for the elderly in any recreational space. 4. Results and Analysis 4.1. Evaluation and Measurement Results Using the above hypothesis and methods described in Section 3.3.3, we obtained the measurement results for the three evaluation indices in each recreation space of Ziyang Park (Table 1). Table 1. Landscape viewability, total view ratio, and water view ratio for the elderly in each recreation space of Ziyang Park. Recreation Space ELV (%) ETVR (%) EWVR (%) Recreation Space ELV (%) ETVR (%) EWVR (%) Wood bench 0 Wood bench 1 Wood bench 2 Wood bench 3 Stone bench 4 Stone bench 5 Xinghe teahouse 6 Cultural corridor 7 45.5 72.7 36.4 36.4 9.1 9.1 72.7 63.6 15.8 21.6 27.4 20.4 8.2 7.3 26.3 30.8 49.7 51.3 39.5 21.6 0.0 0.0 62.0 60.6 Wood bench 8 Wood bench 9 Zhizhi pavilion 10 Miaowan teahouse 11 Zhulu pavilion 12 Meihua Pavilion 13 Daohua Pavilion 14 Hydrophilic platform 15 53.5 45.5 72.7 36.4 18.2 36.4 0.0 90.9 23.9 15.7 29.4 17.0 19.9 25.4 13.5 25.6 63.8 56.2 60.7 55.0 0.0 27.3 0.0 51.3 Based on the above table and Equation (1), we obtained the elderly suitability evaluation results of the visual landscape in each recreation space of Ziyang Park (Table 2). Descriptive statistics show that the elderly suitability mean value was small (0.31), the minimum value (0.07) was close to 0, the maximum value (0.52) was small, and the standard deviation was small (0.151). This indicates that the visual landscape of Ziyang Park’s recreation spaces is not suitable for the elderly and is not able to meet the visual perception demands of the majority of the elderly. Sustainability 2021, 13, x FOR PEER REVIEW Sustainability 2021, 13, 6443 8 of 16 8 of 17 Table 2. Elderly suitability of the visual landscape in each recreation space of Ziyang Park. Table 2. Elderly suitability of the visual landscape in each recreation space of Ziyang Park. Recreation Space ESVL Recreation Space ESVL Recreation Space Recreation Space ESVL Recreation Space ESVL Recreation Space9 Daohua pavilion 14 0.07 Wood bench 3 0.26 Wood bench 0.07 Daohua pavilion Wood bench 3 Wood bench Stone bench145 0.07 Miaowan teahouse 11 0.26 0.29 Wood bench9 2 0.07 0.29 Stone bench 5 Wood bench 2 Miaowan teahouse 11 Stonebench bench 0.08 Meihua Pavilion 0.30 Wood bench8 8 0.08 Stone 4 4 Meihua Pavilion 13 13 0.30 Wood bench 0.17 0.31 Zhulu pavilion 12 Wood bench 0 Wood bench 1 Zhulu pavilion 12 0.17 Wood bench 0 0.31 Wood bench 1 ESVL Recreation Space ESVL ESVL Recreation Space ESVL 0.31 Cultural corridor 7 0.46 0.31 0.46 Cultural corridor 76 0.32 Xinghe teahouse 0.47 0.32 0.47 Xinghe teahouse 6 0.40 Zhizhi pavilion 10 0.49 0.40 0.49 Zhizhi pavilion 10 0.44 0.52 Hydrophilic platform 15 0.44 Hydrophilic platform 15 0.52 4.2. 4.2. Spatial Spatial Distribution DistributionCharacteristics Characteristicsand andCauses CausesofofElderly ElderlySuitability SuitabilityofofVisual VisualLandscape Landscape Based on the above evaluation results, by overlaying the park tin surface, Based on the above evaluation results, by overlaying the park tin surface,recreation recreation points, and main walkway layers in ArcMap 10.4, as well as using the natural points, and main walkway layers in ArcMap 10.4, as well as using the natural breaks breaks method method to torender renderthe thepark parkrecreational recreationalpoint pointelements elementswith withdifferent differentsymbols symbolsaccording according to tothe theelderly elderlysuitability suitabilityvalue valueof oftheir theirvisual visuallandscapes, landscapes,we wecreated createdaaspatial spatialdistribution distribution map of the elderly suitability of the visual landscape for the recreation spaces of Ziyang map of the elderly suitability of the visual landscape for the recreation spaces of Ziyang Park (Figure 3). Park (Figure 3). Figure3.3.Spatial Spatialdistribution distributionofofthe theelderly elderly suitability visual landscape recreation Figure suitability of of thethe visual landscape forfor thethe recreation spaces spaces of Ziyang Park. of Ziyang Park. Figure 33 divides divides all Ziyang Park into three types. The The spaFigure all of ofthe therecreation recreationspaces spacesinin Ziyang Park into three types. tial distribution indicates that the elderly suitability of the visual landscape in the recreaspatial distribution indicates that the elderly suitability of the visual landscape in the tional spacesspaces of Ziyang Park gradually decreases from the interior to theto exterior. More recreational of Ziyang Park gradually decreases from the interior the exterior. specifically, Figure 3 indicates that the elderly suitability level of the visual landscape of More specifically, Figure 3 indicates that the elderly suitability level of the visual landscape recreation at the the main mainentrance entrance of recreationspace spacetype typeI Iisisthe thelowest, lowest,and andthat thatitit is is mainly mainly distributed distributed at andthe theoverlook overlookareas areasofofthe thepark, park,which which are stone bench 4, stone bench 5, Daohua Paviland are stone bench 4, stone bench 5, Daohua Pavilion 14, and Zhulu Pavilion 15. The elderly suitability level of the visual landscape of recreation Sustainability 2021, 13, 6443 9 of 17 space type II is medium, which is mainly distributed on both sides of the park’s main walkway, as well as in low-lying areas. The elderly suitability level of the visual landscape of recreation space type III is the highest, which is mainly distributed in the center of the park and the higher elevation section of the main walkway (such as wood bench 1 and hydrophilic platform 15); most of these areas are characteristic pavilions (such as Zhizhi Pavilion, Xinghe teahouse, and the cultural corridor, among others). Based on the above findings, the comprehensive analysis suggests that the above spatial distribution characteristics may be caused by differences in the location zoning type, surface elevation, road slope, and the characteristics of each recreation space. First, the main entrance of the park is an important channel for users to enter and leave the park; therefore, the recreation space at this location is generally not used by tourists to watch or rest for an extended period. The overlook points of the park are generally distributed at the highest peak on the outer edge of the hill, which are mainly for tourists to enjoy the park’s exterior scenery. As a result, the number of park landscapes and the area of the visual field that elderly users can see when sitting in the recreation space of these two sub-areas are generally few or small. Thus, the elderly suitability level of the visual landscape of recreation space type I is the lowest. Second, if tourists are in low-lying areas, the lines of sight are easily blocked by hills or buildings. Therefore, although recreation space type II is closer to the center of the park than recreation space type I, the elderly suitability of its visual landscape can only reach a medium level. Third, the park center is generally open, often acting as the landscape center, such that the recreation space here generally has certain characteristics, usually characterized by a better observation perspective and a wider field of vision. In addition, some of the recreation spaces in space type III are located in the high slope section of the main walkway, so that they also provide a good perspective and broader vision. For these two reasons, the visual landscape of recreation space type III is the highest and most suitable for the elderly. 4.3. Aging Optimization of Ziyang Park’s Recreational Space Layout As mentioned above, the park’s main entrance is generally not suitable for resting, whereas the park overlook points are generally designed to enjoy the exterior scenery of the park. Therefore, we dismissed the layout optimization of recreation space type I, instead selecting recreation space type II (i.e., recreation spaces on both sides of the main walkway) for layout optimization. Moreover, the established objective was to make the new recreation space layout of Ziyang Park more conducive for the elderly to obtain a superior visual landscape of the park. 4.3.1. Rules and Restrictions According to the above evaluation results and the optimization goal, we established several optimization rules and restrictions based on the geographical elevation, road slope, adjacent distance, and plot area as the investigation factors referring to the classification standard of building slopes [48]. (1) (2) (3) Newly built recreation spaces should be located in plots with a higher elevation or road sections with a higher slope. For each section of the main walkway, if the road slope of a section is high (such as the medium and steep slope sections with a slope >6◦ ) and the existing recreation spaces of this section are scarce, priority should be given to adding small-scale recreation spaces (such as single seats or benches). For plots adjacent to the main walkway, if the plot has a high elevation, low slope (such as flat, gentle, and medium slope plots with slopes <10◦ ), and is sufficiently open (area >100 m2 ), priority should be given to adding a large-scale recreational space (such as a characteristic cool pavilion, chess board, or card field) in such plots. Sustainability 2021, 13, 6443 10 of 17 Sustainability 2021, 13, x FOR PEER REVIEW 10 of 16 4.3.2. Sample Plot Selection and Analysis (1) Sample plot selection of small-scale recreation space based on road slope analysis 4.3.2. Sample Plot Selection and Analysis According to the above rules and restrictions, to measure the road slope of each section Sample plot selection of small-scale recreation based on road slope analysis of the(1) main walkway as accurately as possible, we first space used the editor tool in ArcMap to According towalkway the abovefrom rulesthe and restrictions, road slope of segment the main south entrancetoofmeasure the parkthe at an interval of each 10 msecto tion of72 thesections main walkway accurately as m). possible, we first usedequation the editor(2) tool ArcMap obtain (the lastas section is 9.29 Second, we used toin calculate to segment the main walkway from the entrance of the at anvalue interval of 104). m the slope of each section and rendered allsouth sections according to park the slope (Figure ◦ to obtain 72 sections (the last section is 9.29 m). Second, we used equation (2) to calculate Finally, we extracted the high-altitude endpoints of all road sections with a slope value >6 , the used slopethem of each and plots rendered all sections according to the slope (Figure 4). and as section the sample in which small-scale recreational spacesvalue can be added, i.e., the 11we nodes (v1–v11) Figure 4. endpoints of all road sections with a slope value Finally, extracted the in high-altitude >6°, and used them as the sample plots which small-scale recreational ! spaces can be rin   added, i.e., the 11 nodes (v1–v11) in Figure 4. A = sin−1 | FH − TH |/ (2) ( FX − TX )2 + ( FY − TY )2 −1 𝐴 = sin (|𝐹𝐻 − 𝑇𝐻|⁄√((𝐹𝑋 − 𝑇𝑋)2 + (𝐹𝑌 − 𝑇𝑌)2 )) (2) where the elevation elevationvalues valuesofofthe thestarting startingand and terminal nodes whereFH FHand and TH TH represent the terminal nodes of of the the road section, respectively; represent X coordinates of the starting road section, respectively; FXFX andand TXTX represent thethe X coordinates of the starting andand terterminal nodesofofthe theroad roadsection, section,respectively; respectively; and and FY FY and TY minal nodes TY represent representthe theYYcoordinates coordinates ofofthe thestarting startingand andterminal terminalnodes nodesofofthe theroad roadsection, section,respectively. respectively. Figure4.4.Rendering Renderingofofthe themain mainwalkway walkwayslope slopeand andsample sample plot selection small-scale recreaFigure plot selection of of small-scale recreational tional spaces. we also conducted experiments at intervals and3030m; m;however, however, through spaces. Note: Note: we also conducted experiments at intervals of of 2020 and comparisons,we wefound found that calculation results using 10intervals m intervals were closer to field the field comparisons, that thethe calculation results using 10 m were closer to the survey. survey. (2) Sample plot selection of large-scale recreational spaces based on surface slope analysis (2) adjacent Sample plot of walkway large-scale recreational spaces on surface slope The areasselection of the main were the optimal placesbased for large-scale elderly analysis spaces. Considering that steep terrain will yield many difficulties and costs for recreational The adjacent areasgentle of theslopes main should walkway thefor optimal for large-scale elconstruction, areas with be were selected the siteplaces if possible. Combined derlythe recreational spaces. Considering steep terrain will yield many difficulties and with approximate distance between that the elderly activity areas and the main walkway costs forfrom construction, areas with gentle slopes should be selected the within site if possible. obtained the field survey, we first calculated the surface slope offor areas 30 m of Combined with the approximate distance between the elderly activity areas and the the main walkway (Figure 5). We then selected a total of 10 sample plots suitable formain the walkway obtained from the fieldrecreational survey, wespaces first calculated the surface slope of “the areas construction of large-scale elderly by combining two conditions: 2 ” and “the surface slope should be <10◦ ” (Figure 6). area of a30 single be >100 m within m ofplot the should main walkway (Figure 5). We then selected a total of 10 sample plots suitable for the construction of large-scale elderly recreational spaces by combining two conditions: “the area of a single plot should be >100 m2” and “the surface slope should be <10°” (Figure 6). Sustainability 2021, 13, 6443 Sustainability x FOR PEER REVIEW Sustainability 2021,2021, 13, x13, FOR PEER REVIEW 11 of 11 of 17 16 11 of 16 Figure 5. slope analysis of areas within 30 m of main walkway. Figure 5. Surface Surface slope analysis of the the areas within 30of mthe ofthe the main walkway. Figure 5. Surface slope analysis of the areas within 30 m main walkway. Figure 6. Sample plot selection of large-scale recreational spaces. Figure 6. Sample plotplot selection of large-scale recreational spaces. Figure 6. Sample selection of large-scale recreational spaces. (3) Elderly Elderly Suitability Analysis ofthe the Visual Landscape ofSample Sample Plots Suitability Analysis of Visual Landscape Plots (3) (3) Elderly Suitability Analysis of the Visual Landscape of of Sample Plots Using the same evaluation and analysis method as above, the elderly suitability of Using same evaluationand andanalysis analysismethod methodas as above, above, the elderly ofofthe Using thethe same evaluation elderly suitability suitability the visual landscape in the 21 sample plots was obtained (Tables 3 and 4). thevisual visuallandscape landscapeininthe the21 21sample sampleplots plotswas wasobtained obtained(Tables (Tables3 3and and4).4). Table Elderly suitability ofthe the visual landscape small-scale sample plots. Table 3.3.Elderly suitability of visual landscape ininsmall-scale sample plots. Table 3. Elderly suitability of the visual landscape in small-scale sample plots. Sample Sample Plot Sample PlotPlot V4 V4 V4 V5 V5V5 V7V7 V7 V6V6 V6 V2V2 V2 V3 V3 V3 ESVL ESVL ESVL 0.491 0.491 0.491 0.454 0.454 0.454 0.350 0.350 0.350 0.337 0.337 0.337 0.280 0.280 0.280 0.258 0.258 0.258 Sample Sample Plot Sample PlotPlot V1 V1 V1 V10 V10 V10 V11V11 V11 V9 V9 V9 V8 V8 V8 Average Average Average ESVL ESVL ESVL 0.187 0.187 0.187 0.148 0.148 0.148 0.110 0.110 0.110 0.109 0.109 0.109 0.051 0.051 0.051 0.252 0.252 0.252 Table 4. Elderly suitability of the visual landscape in large-scale sample plots. Table 4. Elderly suitability of the visual landscape in large-scale sample plots. Sample Sample PlotPlot L2 L2 L10L10 L8 L8 ESVL ESVL 0.477 0.477 0.465 0.465 0.444 0.444 Sample Sample PlotPlot L7 L7 L4 L4 L5 L5 ESVL ESVL 0.346 0.346 0.345 0.345 0.334 0.334 Sustainability 2021, 13, x FOR PEER REVIEW Sustainability 2021, 13, 6443 12 of 16 L1 L3 L9 0.402 0.398 0.388 L6 Average 0.22812 of 17 0.383 Table 4. Elderly suitability of the visual landscape in large-scale sample plots. From Tables 3 and 4, we found that only V4, V5, V6, and V7 in the small-scale sample Plotthe existing mean ESVL(0.31), whereasSample ESVL plotsSample exceeded only L6Plot in the large-scale sample plots was slightly L2 lower than the existing 0.477 mean. L7 0.346 L10 0.465 L4 0.345 L8 0.444 L5 0.334 4.3.3. Optimization Suggestions L1 0.402 Park's Recreational L6Space Layout for the 0.228 For optimizing the of Ziyang elderly the L3 0.398 sample plots with an elderly suitability of the visual landscape higher than-the existing L9 0.388 Average 0.383 mean should be selected. However, based on the analysis results presented in the previous section, adding or adjusting large-scale recreational spaces may more easily improve the From Tables 3 and 4, we found that onlyofV4, V5, V6, and V7 in the small-scale sample elderly suitability of the visual landscape Ziyang Park’s recreation spaces. Therefore, plots exceeded the existing mean (0.31), whereas only L6 in the large-scale sample plots combined with the classification rendering in ArcMap (Figure 7), we suggest the followwas ing:slightly lower than the existing mean. (1) First, priority should be given to the addition of large-scale recreational spaces at 4.3.3. Optimization Suggestions L1, L2, L3, L4, L5, L7, L8, L9, and L10, with a preferred order as follows: L2 > L10 > L8 > the>of Park’saccording Recreational Space Layout for the elderly the L1 >For L3 >optimizing L9 > L7 > L4 L5.Ziyang In addition, to the comparison of adjacent spaces, sample plots with an elderly suitability of the visual landscape higher than the existing we also suggest that the existing "Miaowan teahouse" recreational space should be admean justedshould to the be L3selected. location.However, based on the analysis results presented in the previous section, adding or adjusting large-scale recreational spaces may more easily also improve the (2) Second, while adding large-scale recreational spaces, we should consider elderly the visual landscape of Ziyang Park’s recreation spaces. addingsuitability small-scaleofrecreational spaces at V4, V5, V6, V7, with a preferred orderTherefore, as follows: combined with the classification rendering in ArcMap (Figure 7), we suggest the following: V4 > V5 > V7 > V6. Figure7.7.Elderly Elderlysuitability suitabilityclassification classificationofofthe thevisual visuallandscape landscapeofofthe thesample sampleplots. plots. Figure 5. Discussion and Conclusions (1) First, priority should be given to the addition of large-scale recreational spaces at L1, L2,Elderly L3, L4,evaluations L5, L7, L8, L9, with a of preferred orderlargely as follows: L2 > L10 > L8 >perL1 onand the L10, suitability park spaces depends on visual >ception. L3 > L9 However, > L7 > L4 >research L5. In addition, according to the comparison of adjacent spaces, we to date has not discussed how to evaluate and optimize the also suggestof that the existing "Miaowan teahouse" recreational space should be adjusted to suitability parks for the elderly from the perspective of vision or visual landscapes. the L3 location. Based on the influence of the park recreational space layout on the visual landscape for (2) Second, while adding large-scale recreational spaces, we should also consider adding small-scale recreational spaces at V4, V5, V6, V7, with a preferred order as follows: V4 > V5 > V7 > V6. Sustainability 2021, 13, 6443 13 of 17 5. Discussion and Conclusions Elderly evaluations on the suitability of park spaces largely depends on visual perception. However, research to date has not discussed how to evaluate and optimize the suitability of parks for the elderly from the perspective of vision or visual landscapes. Based on the influence of the park recreational space layout on the visual landscape for the elderly and their associated needs for visual landscape suitability, this paper constructed an evaluation index system for the elderly suitability of the visual landscape of park recreational spaces. Further, a method was proposed to achieve the appropriate layout of such spaces based on the evaluation and analysis of the visual landscape from the elderly perspective. The main conclusions are as follows: (1) (2) (3) The elderly suitability of the visual landscape of the recreational spaces in the case park was not only low overall, but also gradually decreased from the interior to the exterior of the park. The above spatial distribution of the elderly suitability of the visual landscape in the case recreational spaces may be caused by the location zoning type, surface elevation, road slope, and the characteristics of each recreational space. The introduction of GIS can transform the original abstract evaluation of elderly suitability of the visual landscape into quantitative data and visual graphics. This is helpful for guiding the development of park features for the elderly in an intuitive and concise manner, and it could promote the sustainable development of urban societies. Moreover, the combined application of the Python Scripting technology also significantly improved the efficiency and accuracy of our evaluation and optimization. This study proves that the elderly suitability evaluation and analysis of the visual landscape of park recreational spaces can really provide a positive enlightenment for the aging construction of urban parks. However, in practical applications, the influence of other non-spatial factors on the visual landscape should also be considered, such as the specific form of a recreational space and the time node of the elderly’s activities. Located in the western part of the case park, the cultural corridor is the most commonly used and favorite recreational space of the elderly; its specific form is a strip from the east to the west. Our results indicate that the elderly suitability of the visual landscape of this space is highest. However, in spring and summer, owing to the lush flowers and trees along the lakeside, the number of landscapes, field of vision, and water area that the elderly can enjoy in this space is significantly less than the theoretical value. Therefore, in the specific optimization practice, considering such non-spatial factors should be combined with field investigations, with appropriate adjustments or supplements to the theoretical optimization scheme. The results of this study can also be applied adapt other spaces in the park, such as park fitness spaces, for the aging population. However, in the process of visiting the park, elderly tourists generally expect to have a step-by-step visual experience of changing scenery. Therefore, the landscape repetition rate may be a key point that should be considered in future research. In planning and design, if the landscape repetition rate of adjacent recreational spaces can be effectively reduced, we can more likely create an increasingly suitable recreational space for the elderly. Although Li et al. [26] have considered the landscape repetition rate problem in terms of the viewshed analysis of parks, whether we can use their research to achieve a solution still remains a future challenge. At present, as there is a lack of research on the elderly suitability of visual landscapes, there is no comparable basis or reasonable range for the elderly suitability of visual landscapes in relevant spaces. Compared with previous studies (especially those supported by GIS) [5,9,16,20,23,40], despite their identical intuitive guiding significance for planning and transformation, this study has two innovations. First, the concept of elderly suitability of the visual landscape and its evaluation index system were proposed. This provides a new research perspective to evaluate and optimize the elderly suitability of parks. Second, Python Scripting for ArcGIS was used to evaluate the elderly suitability and analyze the visual landscape efficiently, which provides a reference method for related research. How- Sustainability 2021, 13, 6443 14 of 17 ever, this study still has limitations. As the selected park is a small-scale community park, the relevant geographic elevation could only be measured manually; therefore, certain deviations in the data inevitably occurred. In addition to the limited visual distance, other visual features of the elderly should be considered in future studies. Author Contributions: Conceptualization, W.Y. and B.S.; methodology, W.Y., H.H., and B.S.; software, W.Y. and H.H.; validation, W.Y. and B.S.; formal analysis, W.Y. and B.S.; resources, W.Y.; data curation, W.Y.; writing—original draft preparation, W.Y.; writing—review and editing, B.S. and H.H.; supervision, W.Y. and B.S.; project administration, W.Y.; funding acquisition, W.Y. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Project of Humanities and Social Sciences Research Planning Foundation of the Chinese Ministry of Education (No. 17YJAZH113). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicly available due to ongoing research in this field. Acknowledgments: We would like to thank respected editors and anonymous reviewers for their insightful suggestions and comments in improving the quality of the paper. Conflicts of Interest: The authors declare no conflict of interest. Appendix A The following key codes were customized by Python Scripting for ArcGIS, in which the sentences beginning with # are the comments on the codes. Appendix A.1. Calculate the Line Network of Sight from Each Recreation Point to Each Landscape Point, so as to Obtain the Visible Landscape Number and the Landscape Viewability for the Elderly in Each Recreation Space import arcpy arcpy.env.workspace = ‘F:\ZY20\1004’ # Set environments arcpy.ddd.ConstructSightLines(“zqd_16”, “jgd_12”, “z16_j12_sls.shp”, “Obs_Z”, “Tar_Z”,”<None>”, 1,”OUTPUT_THE_DIRECTION”) # zqd_16 and jgd_12 are the shplayer of recreation points and landscape points, respectively, and z16_j12_sls is the line network of sight. arcpy.ddd.Intervisibility(“z16_j12_sls”, “zybetin10”, “Visibility”) Appendix A.2. Separate zqd_16.shp into Several Independent Shplayers, and then Calculate the Total Viewshed Area for the Elderly in Each Recreation Space by the Viewshed Function of ArcPy, so as to Calculate the Total View Ratio for the Elderly in Each Recreation Space import arcpy,math,sys,os from arcpy import env from arcpy.sa import * reload(sys) in_feature = “F:/ZY20/Oct/AvShed/zqd_16.shp “ split_field = “Name” field_data_list = [] with arcpy.da.SearchCursor(in_feature,split_field) as cursor: for row in cursor: if row [0] not in field_data_list: field_data_list.append(row[0]) Sustainability 2021, 13, 6443 15 of 17 out_folderpath = “F:/ZY20/Oct/AvShed/dpartZ16” # Set the storage path for the independent shplayers for select_data in field_data_list: arcpy.Select_analysis(in_feature,out_folderpath+’/’+select_data+’.shp’,split_field+” = “+”’”+select_data+”’”) # Separate outViewshed = Viewshed(“union10_ras.tif”,select_data+’.shp’,1) # Calculate the total viewshed area outViewshed.save(“F:/ZY20/Oct/AvShed/AvShed.gdb/V”+select_data) # Store the total viewshed area, and “V”+select_data is the total viewshed area of a recreation point. Appendix A.3. Calculate the Water Viewshed Area for the Elderly in Each Recreation Space by the ExtractByMask Function of ArcPy, so as to Calculate the Water View Ratio for the Elderly in Each Recreation Space import arcpy from arcpy import env from arcpy.sa import * in_feature = “F:/ZY20/Oct/AWvShed/zqd_16.shp “ split_field = “Name” WvShName_list = [] with arcpy.da.SearchCursor(in_feature,split_field) as cursor: for row in cursor: if row[0] not in WvShName_list: WvShName_list.append(row[0]) env.workspace = “F:/ZY20/Oct/AWvShed/WvShed.gdb” for select_data in WvShName_list: outExtractByMask = ExtractByMask(“V”+select_data, “HmDgm”) # “Hmdgm” is the park lake surface. outExtractByMask.save(“F:/ZY20/Oct/AWvShed/WvShed.gdb/Wv”+select_data) # “Wv”+select_data is the water viewshed area of a recreation point. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Yung, E.H.K.; Ho, W.K.O.; Chan, E.H.W. 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RJOAS, 3(75), March 2018 DOI https://doi.org/10.18551/rjoas.2018-03.20 STUDY OF LOCAL GOVERNMENT PROBLEMS IN IMPLEMENTATION OF DEVELOPMENT BUDGET POLICY PLANNING IN ECONOMIC SECTOR OF INDONESIA Silalahi Engelbertha E.*, Aba Fransiskus X Lara, Lydia Ni Luh Gde Faculty of Economics and Business, Atma Jaya Catholic University, Indonesia *E-mail: engelberthasilalahi1972@gmail.com ABSTRACT Purpose of this study is to provide recommendations for development budget planning in leading sectors of the economy. Preparation of policy planning of Regional Revenue and Expenditure Budget by using this performance-based budget system prioritizes efforts to achieve the allocation of output work plan and input costs. The projection shows, GRDP in Jakarta of Indonesia increased from Indonesia Rupiah (IDR) 543.02 trillion in 2016 to IDR. 565.42 trillion in 2017. Furthermore GRDP numbers continue to increase and reach the figure of IDR 659.32 trillion in 2021. Similarly, regional expenditure, the projection also shows that in 2017 regional expenditure has decreased to IDR 39.37 trillion (18.07%) of regional revenue in 2016 amounting to IDR. 48.06 trillion. KEY WORDS Budget, policy, development, planning, local government, economic sector. The study of the implementation of economic development budget policy can not be separated from the impact of planning that must be seen as an integral part in every process both in making and implementation of the decision. As a consequence, the policy of planning will be meaningless unless decentralization is followed in its implementation (Conyers et al, 1990). According to Austen and Banks, (1988) Decentralization itself is the responsibility for the planning and or alternative implementation of the balance of power between the central and regional governments in formulating the economic development budget policy on what which will be faced for development in the present and future. In other words, the implementation of a decentralized development budget planning policy is a political process that needs to be further analyzed. Implementation of local financial policies should be harmoniously in support of development programs to achieve substantial regional economic growth (Aba, Yussof, Saidatulakmal.; 2015). Government revenue and expenditure budgets need to be laid out in a budget system capable of improving governance both in general governmental duties and development tasks (Foremny, et al, 2014). Some of the causes that make the budget in Jakarta Indonesia hampered, the result of the delay is caused by (1). The organizational stability is not conducive for the Regional Device Work Unit (RDWU) to show good performance. Even RDWU, tend to be very careful to make a breakthrough through a program. (2). Government steps in Jakarta Indonesia using e-budgeting as a budgeting system is also still immature. This makes RDW difficult to apply. (3). To be imposed ebudgeting, ultimately accessing the opening of game gaps in auctions made via e-budgeting (Schofield and Sened., 2006). Routine and development budget in Jakarta Indonesia needs to be more synchronized and consolidated according to budget planning that is continuous, gradual and increasing with maximum orientation of the results. Based on the above point of view, the problem of local government in absorbing Regional Budget of Jakarta is still very low. Based on reports from the regional finance and asset management board, the absorption of the budget only reached 13.86 percent or IDR. 8.03 trillion from the total IDR.67.1 trillion. The lack of budget absorption is considered to have implications for the low cost of infrastructure development for communities in Jakarta Indonesia. In that context, Jakarta Indonesia still needs a high cost to build an adequate public infrastructure. Moreover, people need the realization of the 179 RJOAS, 3(75), March 2018 maximum budget to finance development in Jakarta Indonesia. This provides a role in the economic base of income as well as the position of each economic activity for the Regional Revenue and Expenditure Budget (RREB) in the absorption of the budget for the acceleration of economic improvement and public services in Jakarta Indonesia (Bararuallo, and Aba., 2017). METHODS OF RESEARCH Analysis of Structure and Growth of RREB (Adiab, et al., 2007) is the RREB Structure analyzed by looking at the contribution of revenue sources and expenditure items relative to total RREB in Jakarta Indonesia. According to Baltagi, (2001), revenue growth and expenditure in RREB for eight years (1993-2000) will be calculated using the exponential trend equation (Holt, 1957), and projection analysis from 2017 - 2021. Analysis of RREB Capacity (Albacete, and Lindner, 2013), is to look at the performance of local government revenues and expenditures of Jakarta Indonesia using the ratio of revenue to GRDP and revenue to development budget (Gomes, 2015). RESULT OF STUDY Based on Hunter Blair (2016), the budget is the estimated performance to be achieved over a given period of time expressed in financial size. the budget can be defined as a financial plan in the form of expenditures and sources of income for one year. There is a close relationship between budget, planning and control (Arian and Shamir, 2011). Planning is used to see what action should be taken to achieve a particular goal in the future (Blekesaune .; 2007), while control is more looking backward, determining what actually happened and comparing it with planning. Meanwhile, according to Binzer and Klemmemsen (2005), the budget is a document showing the condition or financial condition of an organization that provides information on income (Alesina and Tabellini, 2007), expenditures, activities and objectives to be achieved. Revenue and Expenditure Budget (RREB) is the state budget prepared every year. Therefore, the RREB has a very important role because it becomes one of the main tools for the welfare of the community. Budgeting is a process or method for preparing a budget (Miekatrien Sterck and Bram Scheers, 2006). Budgeting is a process of translating an activity plan into a financial plan (Weimer and Vining, 2005). In a broader sense, budgeting involves budget preparation, implementation, control, and accountability known as the budget cycle. Budgeting is related to the process of determining the amount of fund allocations for programs and activities. So, it can be concluded that the budget is the driving force and budgeting is the process to prepare the budget. One of the instruments that can be used to analyze the financial capacity of the region is the instrument of GRDP (BPS.; 2017). The instrument provides an indication of the potential and dominant role of the regional economic sector seen from the creation of output and added value as well as final demand (Kuhlmann, 1998). Ratio of RREB to GRDP is to see how far the role of local government in providing public services through government expenditure in RREB (Aba., 2017). Table 1 – Ratio of Regional Revenue and Expenditure Budget / RREB to GRDP in Jakarta Indonesia, 1992-1997 Years 1992 1993 1994 1995 1996 1997 RREB (Million IDR) 1,130,731.3 1,476,521.3 1,847,936.7 2,404,720.8 2,835,833.8 2,981,385.6 GRDP (Million IDR) 16,001,557.0 51,106,389.0 55,505,268.0 60,638,216.0 66,164,802.0 69,543,347.0 Source: Data Analyzed by Researcher. 180 Ratio (%) 7.1 2.9 3.3 4.0 4.3 4.3 RJOAS, 3(75), March 2018 The expenditure ratio on GRDP performance in Jakarta Indonesia showed a declining trend in 1993 (2.9%), then increased annually from 1994 (3.3%) to 1997 (4.3%). Means, the ability of local governments in providing public services is getting better. The proportion of expenditure in the Revenue and Expenditure Budget is an indicator to see the effectiveness of government expenditures on increasing output generated in the regional economy. In other words, the ratio of Revenue and Expenditure Budget to the increasing GRDP shows that the proper allocation of expenditure budget can spur economic development in Jakarta Indonesia. The expenditure ratios on GRDP performance in Jakarta Indonesia showed a downward trend in 1998 (3.1%), then increased in 1999 (6.0%), then decreased annually from 2000 (5.2%) to 2002 (3, 5%). This means, the ability of local governments in providing public services has not improved. The proportion of expenditure in the Revenue and Expenditure Budget is an indicator to see the effectiveness of government expenditures on increasing output generated in the regional economy. In other words, the ratio of Revenue and Expenditure Budget to the decreasing GRDP indicates that the expenditure budget has not been appropriately allocated in order to spur economic development in Jakarta Indonesia. More can be seen in the table below. Table 2 – Ratio of Regional Revenue and Expenditure Budget / RREB to GRDP in Jakarta Indonesia, 1997-2002 Years 1997 1998 1999 2000 2001 2002 RREB (Million IDR) 2,981,385.6 1,802,068.9 3,434,601.1 3,127,364.2 9,274,825.6 8,754,245.8 GRDP (Million IDR) 69,543,347.0 57,380,517.0 57,215,224.0 59,694,418.0 238,656,139.0 250,331,157.0 Ratio (%) 4.3 3.1 6.0 5.2 3.9 3.5 Source: Data Analyzed by Researcher. The expenditure ratio on GRDP performance in Jakarta Indonesia shows an increasing trend every year from 2003 (3.9%) to 2007 (5.2%). This means, the ability of local governments in providing public services is improving. Table 3 – Ratio of Regional Revenue and Expenditure Budget / RREB to GRDP in Jakarta Indonesia, 2002-2007 Years 2002 2003 2004 2005 2006 2007 RREB (Million IDR) 8,754,245.8 10,382,597.1 11,493,273.3 12,435,352.4 15,161,577.7 17,280,823.4 GRDP (Million IDR) 250,331,157.0 263,624,242.0 278,524,823.0 295,270,545.0 312,751,711.0 332,971,255.0 Ratio (%) 3.5 3.9 4.1 4.2 4.8 5.2 Source: Data Analyzed by Researcher. The proportion of expenditure in the Revenue and Expenditure Budget is an indicator to see the effectiveness of government expenditures on increasing output generated in the regional economy. In other words, the ratio of Revenue and Expenditure Budget to the increasing GRDP indicates that the proper allocation of expenditure budget can spur economic development in Jakarta Indonesia. Ratio of expenditure on GRDP performance in Jakarta showed a declining trend in 2008 (4.5%), then increasing every year from 2009 (5,3%) until 2012 (7.0%). This means, the ability of local governments in providing public services is improving. The proportion of expenditure in the Revenue and Expenditure Budget is an indicator to see the effectiveness of government spending on increasing output generated in the regional economy. In other words, the ratio of Revenue and Expenditure Budget to the 181 RJOAS, 3(75), March 2018 increasing GRDP shows that the proper allocation of expenditure budget has been able to spur economic development in Jakarta Indonesia. Table 4 – Ratio of Regional Revenue and Expenditure Budget / RREB to GRDP in Jakarta Indonesia, 2007-2012 Years 2007 2008 2009 2010 2011 2012 RREB (Million IDR) 17,280,823.4 15,956,526.1 19,511,099.4 21,555,447.7 26,423,682.2 31,558,706.9 GRDP (Million IDR) 332,971,255.0 353,723,390.0 371,469,500.0 395,633,574.0 422,121,511.0 449,805,475.0 Ratio (%) 5.2 4.5 5.3 5.4 6.3 7.0 Source: Data Analyzed by Researcher. The expenditure ratio on GRDP performance in Jakarta Indonesia shows an increasing trend in 2013 (8.0%), then declines in 2014 (7.5%), and further increases again in 2015 (8.2%) through 2016 (8.9%). This means, the ability of local governments in providing public services is improving. The proportion of expenditure in the Revenue and Expenditure Budget is an indicator to see the effectiveness of government expenditures on increasing output generated in the regional economy. In other words, the ratio of Revenue and Expenditure Budget to the increasing GRDP shows that the proper allocation of expenditure budget has been able to spur economic development in Jakarta Indonesia. Table 5 – Ratio of Regional Revenue and Expenditure Budget / RREB to GRDP in Jakarta Indonesia, 2012-2016 Years 2012 2013 2014 2015 2016 RREB (Million IDR) 31,558,706.9 38,294,384.9 37,759,773.0 43,031,322.9 48,059,574.8 GRDP (Million IDR) 449,805,475.0 477,285,245.0 504,225,592.0 523,925,770.0 543,020,428.0 Ratio (%) 7.0 8.0 7.5 8.2 8.9 Source: Data Analyzed by Researcher. The budgeting system in Indonesia is reflected in the State Budget. According to Goyal Ashima (2010), the state budget is the government's annual financial plan approved by the House of Representatives, which contains a systematic list and details of state revenue and expenditure plans for one fiscal year (1 January-31 December) act and implemented openly and responsibly to maximize people's prosperity. The consists of state revenues and grants, state expenditures, and financing. State Budget is the main instrument of fiscal policy to direct the national economy and stimulate economic growth. The amount of budget absorption will have an impact on the higher rate of economic growth. The ratio of realized expenditure absorption of Ministries or Institutions to budget realization is a form of indicator of effectiveness of state expenditure. In addition, State Badget policy is expected to respond to the dynamics of the people both related to broad economic development, as well as the life of the people themselves, so that required a flexible fiscal policy. To see the picture of the economy in Jakarta Indonesia in the future can be done by projecting the amounts obtained in the structure of GRDP. Projection Results GRDP in Jakarta Indonesia 5 years (2017 - 2021) increased from IDR. 543.02 trillion in 2016 to IDR. 565.42 trillion in 2017. Furthermore GRDP numbers continue to increase and reach the figure of IDR. 659.32 trillion in 2021. The analysis results show the result of projection of regional and government expenditure in Jakarta Indonesia in 2017 - 2021. Projection data indicate that in 2017 local revenue decreased to IDR. 41.40 trillions (12.91%) from the previous year's revenue of IDR. 47.54 trillion. Regional income figures then increase every year to reach IDR. 49.07 trillion in 2021. 182 RJOAS, 3(75), March 2018 Similarly, regional spending, the projection also shows that in 2017 regional expenditure has decreased to IDR. 39.37 trillion (18.07%) of the previous year's revenues of IDR. 48.06 trillion. Regional expenditure subsequently increase every year to reach IDR. 46.71 trillion by the year 2021. DISCUSSION OF RESULTS Regional economic development efforts face various opposition from within and outside (Bargsted and Kedar., 2009). This situation requires the ability and policy of Jakarta Indonesia government to make the process of accelerating the preparation and implementation of economic development that focus on economic base. The results of the study indicate that the use of development budget in Jakarta Indonesia relies too much on development costs to non-RREB funds. Local government in Jakarta Indonesia has financed development with Corporate Social Responsibility (CSR) funds. Such a breakthrough is even contradictory to the RREB that has been approved because it makes him abandoned and not absorbed maximally. So far, programs conducted using non-budget funds such as CSR, to note, to date there are indeed some infrastructure in Jakarta Indonesia built using CSR funds such as Lenggang Jakarta, the location built for street vendors Monas area. In addition, there is also the procurement of garbage trucks, Integrated Child Friendly Public Space Development, procurement bus city tour level, handling Pluit dam and Rio Ria. Comparative study of the implementation of economic development budget (Armstrong, and Taylor (2000) in collecting data and information on the state of the region and the potential of the economic sectors it possesses as input determination in clarifying the regional economic picture objectively, thus providing the necessary input in determination of local government policy (Rosenthal, 2012) for the creation of innovative economic development systems (Conyers and Hills, 1990). CONCLUSION Based on budget implementation as part of policy implementation, it can be assumed that budget implementation is influenced by interests that focus on political power and decisions. The outcomes of economic development and policy implementation by the Regional Government in Jakarta Indonesia can ultimately be evaluated and assessed for success through the economic measures of budget use that have been achieved according to plan, it is seen through the government's accountability report, , objective community reports as well as reports from the results of scientific research conducted by the community or social institutions that demonstrate the performance of the implementation of regional development. From the discovery shows the trend of budget use for 25 years showed fluctuations in the absorption. This is seen also at the end of the year projection results show, GDP in Jakarta Indonesia increased from IDR. 543.02 trillion in 2016 to IDR. 565.42 trillion in 2017. Furthermore GRDP numbers continue to increase and reach the figure of IDR. 659.32 trillion in 2021. 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ORIGINAL RESEARCH published: 15 July 2015 doi: 10.3389/fpsyg.2015.00981 Arousal, valence, and the uncanny valley: psychophysiological and self-report findings Marcus Cheetham1,2*† , Lingdan Wu 3,4† , Paul Pauli4 and Lutz Jancke1 1 Department of Neuropsychology, University of Zurich, Zurich, Switzerland, 2 Department of Psychology, Nungin University, Seoul, South Korea, 3 Swiss Centre for Affective Sciences, University of Geneva, Geneva, Switzerland, 4 Department of Psychology, University of Wurzburg, Wurzburg, Germany Edited by: Eddy J. Davelaar, Birkbeck, University of London, UK Reviewed by: Francesca M. M. Citron, Lancaster University, UK Christian Becker-Asano, Albert-Ludwigs-Universität Freiburg, Germany *Correspondence: Marcus Cheetham, Department of Neuropsychology, University of Zurich, Binzmühlestrasse 14/Box 25, CH-8050 Zürich, Switzerland m.cheetham@psychologie.uzh.ch † These authors share first authorship. Specialty section: This article was submitted to Cognitive Science, a section of the journal Frontiers in Psychology Received: 15 November 2014 Accepted: 29 June 2015 Published: 15 July 2015 Citation: Cheetham M, Wu L, Pauli P and Jancke L (2015) Arousal, valence, and the uncanny valley: psychophysiological and self-report findings. Front. Psychol. 6:981. doi: 10.3389/fpsyg.2015.00981 The main prediction of the Uncanny Valley Hypothesis (UVH) is that observation of humanlike characters that are difficult to distinguish from the human counterpart will evoke a state of negative affect. Well-established electrophysiological [late positive potential (LPP) and facial electromyography (EMG)] and self-report [Self-Assessment Manikin (SAM)] indices of valence and arousal, i.e., the primary orthogonal dimensions of affective experience, were used to test this prediction by examining affective experience in response to categorically ambiguous compared with unambiguous avatar and human faces (N = 30). LPP and EMG provided direct psychophysiological indices of affective state during passive observation and the SAM provided self-reported indices of affective state during explicit cognitive evaluation of static facial stimuli. The faces were drawn from well-controlled morph continua representing the UVH’ dimension of human likeness (DHL). The results provide no support for the notion that category ambiguity along the DHL is specifically associated with enhanced experience of negative affect. On the contrary, the LPP and SAM-based measures of arousal and valence indicated a general increase in negative affective state (i.e., enhanced arousal and negative valence) with greater morph distance from the human end of the DHL. A second sample (N = 30) produced the same finding, using an ad hoc self-rating scale of feelings of familiarity, i.e., an oft-used measure of affective experience along the UVH’ familiarity dimension. In conclusion, this multi-method approach using well-validated psychophysiological and self-rating indices of arousal and valence rejects – for passive observation and for explicit affective evaluation of static faces – the main prediction of the UVH. Keywords: valence, arousal, uncanny valley hypothesis, familiarity, EMG, EEG, LPP Introduction The longstanding Uncanny Valley Hypothesis (UVH) predicts that difficulty distinguishing a realistic humanlike character or object (e.g., robot, prosthetic hand) from its human counterpart will evoke an unpleasant affective state (Mori, 1970; Figure 1). Mori suggests that this state is characterized by a sense of strangeness and personal disquiet and, when experienced more intensely, by revulsion and disgust. Attention to the originally untested UVH has been spurred Frontiers in Psychology | www.frontiersin.org 1 July 2015 | Volume 6 | Article 981 Cheetham et al. Uncanny hypothesis and effect the positive and negative character of affective experience as affinity. An alternative approach to investigating affective experience of human and humanlike entities is to consider affective experience in terms of its psychologically well-validated components. Mori (1970) uses illustrative instances of feelings and affective evaluations to describe his understanding of shinwakan. Based on these instances, shinwakan could well be considered in terms of the constructs valence and arousal because these are intrinsic to all of his examples (cf. Davitz, 1969). These are also intrinsic to the affective dimensions (e.g., likeability, pleasantness, feelings of familiarity) typically used to examine the UVH and to all of the early theoretical accounts of uncanny experience (e.g., MacDorman, 2005), and they appear to be relevant for the definition of uncanny feelings in terms of specific emotions (cf. Russell, 1980; Ho et al., 2008). Valence refers to the pleasant-to-unpleasant quality (hedonic tone) and arousal to the low-to-high degree of excitement of affective experience (Barrett and Russell, 1999; Kensinger and Corkin, 2004). Given that valence and arousal form the primary orthogonal dimensions of affective experience (Schlossberg, 1954; Russell, 1980, 2003; Yik et al., 1999; Posner et al., 2005), that emotional states can be defined in the emotional space determined by these two dimensions (Bradley and Lang, 1994; Barrett and Russell, 1999), and that the principal variance in the meaning of emotional states can be explained by valence and arousal (Osgood et al., 1957; Mehrabian and Russell, 1974; Smith and Ellsworth, 1985), it is likely that indicators of valence and arousal will provide a sound basis for examining affective experience of variously humanlike entities along the DHL and the notion of the uncanny effect. A second reason for inconsistent findings is likely to relate to the more or less exclusive reliance in uncanny-related research on ad hoc developed self-rating scales to assess affective experience (e.g., Hanson, 2006; MacDorman, 2006; Green et al., 2008; MacDorman et al., 2009a,b; Seyama and Nagayama, 2009; Looser and Wheatley, 2010; Tinwell et al., 2010). Ad hoc self-rating scales have found favor in uncanny research because they are inexpensive and easy to administer (see, e.g., Ho et al., 2008). But the psychometric validity and reliability of these measures as indicators of shinwakan, or of the affective experience that the notion of shinwakan is thought to capture, has not been demonstrated (but for steps toward construct validation, see Ho and MacDorman, 2010). This makes the interpretation and synthesis of the research findings to date difficult. As an alternative to ad hoc scales, and in keeping with the foregoing considerations on arousal and valence, well-validated measures such as the Self-Assessment Manikin (SAM, e.g., Lang, 1985; Lang et al., 1997; Bradley and Lang, 2007) could be used. The SAM is a pictorial assessment technique for self-rated measurement of valence and arousal. As a non-verbal and largely culture free measure (Morris et al., 1993; Bradley et al., 1994), it would be useful – given the preceding concerns about the ambiguous concept shinwakan – for application in uncannyrelated research. Though a valuable source of information, selfratings of affective experience (e.g., of pleasantness, comfort level, eeriness, familiarity, likability, valence, or arousal) effectively FIGURE 1 | Illustration of the Uncanny Valley Hypothesis. The main prediction of the Uncanny Valley Hypothesis is that observation of highly humanlike characters and objects (depicted along the dimension of human likeness) will evoke a sharp negative peak in affective experience (depicted along the familiarity dimension). This negative peak, referred to as the uncanny valley, is characterized by feelings of strangeness and disquiet. These feelings are suggested to be stronger for dynamic stimuli. The valley occurs at the point along the dimension of human likeness at which category membership as human or non-human is highly ambiguous (illustration adapted from MacDorman, 2005). by recent progress in robotics and computer graphics technologies in the realistic simulation of aspects of human appearance and behavior and, therefore, by interest in understanding the impact of enhanced anthropomorphic realism on affective experience (e.g., Ho and MacDorman, 2010; Yamada et al., 2013). But empirical support for the predicted uncanny effect has been inconsistent (e.g., Hanson, 2006; MacDorman, 2006; Tinwell and Grimshaw, 2009; MacDorman et al., 2013). This has led to the query as to how research should now best proceed (Zlotowski et al., 2013). To move beyond this seeming impasse, some of the reasons for inconsistency in findings and new avenues of approach can be considered. One reason for inconsistency stems from the UVH’ ambiguous definition of the concept shinwakan. Mori coined this Japanese neologism to describe in simple terms the positive and negative character of affective experience in response to variously humanlike objects (Figure 1); the UVH defines these objects along a dimension of human likeness (DHL). Though relating to affective experience, the poor specification of this concept has resulted in much debate and various renderings of its meaning, with the investigation of the UVH and shinwakan in terms of constructs such as pleasantness, comfort level, eeriness, familiarity (i.e., feelings of familiarity vs. strangeness), likability, and empathy (e.g., MacDorman and Ishiguro, 2006; Bartneck et al., 2007; Seyama and Nagayama, 2007, 2009; Green et al., 2008; MacDorman et al., 2009a, 2013; Tinwell and Grimshaw, 2009; Dill et al., 2012; Burleigh et al., 2013). Adding to, or reflecting, the UVH’ conceptual ambiguity, Mori (2012) recently re-termed Frontiers in Psychology | www.frontiersin.org 2 July 2015 | Volume 6 | Article 981 Cheetham et al. Uncanny hypothesis and effect Understanding the human–nonhuman category structure of the DHL provides an approach to testing the ideas underlying the vaguely formulated UVH (Cheetham et al., 2011, 2014; Burleigh et al., 2013; Yamada et al., 2013; Burleigh and Schoenherr, 2015; Ferrey et al., 2015). The UVH makes no explicit reference to perceptual and category processing or to the large body of pertinent literature. But, based on this understanding, Mori’s ideas can be tested by augmenting the UVH with the assumption that the predicted state of negatively valenced affect is most likely to occur at the point of realism along the DHL at which attribution of stimuli to the human or non-human category is subject to greatest categorization ambiguity (i.e., the “valley” in Figure 1). A similar approach has been applied to examining the relationship between the predicted state of negatively valenced affect and the ability to perceptually discriminate between (rather than categorize) morphs along continua representing the DHL (Cheetham et al., 2014). Cheetham et al. (2014) reported a number of effects that, however, provided no support for Mori’s ideas. The aim of this study was to examine affective experience in response to the presentation of highly similar human and humanlike facial stimuli along the DHL. In view of the uncertainty surrounding the conceptual definition and translation of shinwakan, we focused in the main experiment (i.e., the first of two experiments) on the examination of valence and arousal as two of the primary properties of affective experience, using non-verbal measures. The facial EMG measure of the corrugator supercilii muscle and the LPP were used as psychophysiological indices of valence and arousal, respectively. The SAM was used to assess self-reported valence and arousal. The DHL was represented using morphs drawn from carefully controlled continua generated from avatar and human faces. These continua were previously tested to ensure that the cognitive representation of the category structure (i.e., morph location of categorically highly unambiguous avatar and human face exemplars and of categorically most ambiguous faces) was consistent across continua. To test the main prediction of the UVH, we assumed that the face morph associated with greatest category ambiguity along the DHL would evoke greater negatively valenced and more arousing affective experience compared with that evoked by categorically unambiguous avatar and human morphs. In the second experiment, we examined the relationship between the DHL and shinwakan in order to provide, using our stimuli, a general reference of comparison with previous studies that have focused on shinwakan using ad hoc scales. For this, an ad hoc self-rating scale of shinwakan based on the bi-polar dimension of familiarity (i.e., feelings of familiarity vs. strangeness) was used. Familiarity was selected because this rendering of shinwakan has been frequently investigated and because it closely captures the essence of Mori’s description of the uncanny. In keeping with the preceding considerations on category structure, and assuming for the purpose of experimentation that Mori’s conjectures are correct, self-rated experience of familiarity was expected to show that feelings of strangeness would be greater along the DHL for categorically ambiguous morphs compared with categorically unambiguous avatar and human morphs. place the focus of investigation on conscious feelings of emotional state (Barrett, 1996), that is, on the explicit cognitive evaluation of the affect-related properties of the stimuli and of the psychophysiological reactions that these stimuli elicit. It is, however, conceivable that the proposed uncanny effect might also manifest itself in affect-related reactions that escape conscious detection and evaluation (for affect-related neural processes during passive category processing of faces along the DHL, see Cheetham et al., 2011). Self-report measures can be augmented therefore by direct measures of psychophysiological reactions. For the present study, we considered the use of facial electromyography (EMG) and electroencephalography (EEG) to indicate rapid and subliminal changes in emotional state (Wu et al., 2012). Affective experience is associated with psychophysiological indices that correlate differentially with valence and arousal (Cacioppo et al., 1986; Bradley and Vrana, 1993; Lang et al., 1993, 1997; Schupp et al., 2000; Amrhein et al., 2004; Foti and Hajcak, 2009): the EMG measures of the corrugator supercilii muscle and the zygomaticus major muscle are sensitive to negative and positive valence, respectively, and the EEG-based measure of the late positive potential (LPP) to arousal (Cacioppo et al., 1986; Lang et al., 1993, 1997; Cuthbert et al., 2000; Schupp et al., 2000; Dolcos and Cabeza, 2002; Amrhein et al., 2004; Hajcak and Nieuwenhuis, 2006; Foti and Hajcak, 2009; Hajcak et al., 2009; Weinberg et al., 2012; Wu et al., 2012). A further reason for inconsistency in findings has been suggested to relate to the conceptualization and operationalization of the DHL (Cheetham et al., 2011). Mori’s illustration of the UVH uses a single human exemplar to represent the human category (Mori, 1970), as reflected in some empirical and theoretical work (e.g., Ramey, 2005; Tinwell and Grimshaw, 2009). But this conceptualization effectively assumes that there is no variation in physical or psychological similarity space within the human category of the DHL. Examination of perceptual discriminative and category processing along the DHL, operationalized using morph continua to represent a linear dimension of physical similarity space spanning between human and non-human category exemplars, shows that this assumption is incorrect (Cheetham et al., 2011, 2014). The use of linear morph continua to represent the DHL is not new (e.g., Seyama and Nagayama, 2007), but poor control of continua might have contributed to inconsistency in findings. Morph continua have been subject to various experimental confounds that are likely to have systematically biased subjective experience of objects along the DHL (for a critical discussion, see Cheetham and Jancke, 2013). These confounds range from the use of different juxtaposed morph continua to represent the DHL, thus generating perceptual discontinuities along the morph continua (Hanson et al., 2005; MacDorman and Ishiguro, 2006) to morphing noise (e.g., disparities in the alignment of facial features between successive morphs of a continuum), as indicated in a recent study of the effects on subjective experience of category ambiguity (Yamada et al., 2013). Critically, morphing noise is likely to alter the cognitive representation of the human–nonhuman category structure of the DHL and may itself influence subjective experience (Cheetham and Jancke, 2013). Frontiers in Psychology | www.frontiersin.org 3 July 2015 | Volume 6 | Article 981 Cheetham et al. Uncanny hypothesis and effect Materials and Methods presented stimulus as either an avatar or human as quickly and accurately as possible after stimulus onset by pressing one of two response keys. These pilots showed that faces at morph positions M0, M1, M2, and M3 were highly unambiguously assigned to the avatar category, faces at M9, M10, M11, and M12 were highly unambiguously assigned to the human category, and that M6 was most closely associated with greatest ambiguity in categorization judgments (for details and Supplementary Figure S1A, see Supplemental Information 1). Another pilot study (N = 18) was used to judge the facial attractiveness of avatar and human endpoints of all continua before these were morphed. A dependent sample t-test showed that there was no significant difference in attractiveness ratings between the avatar (M = 2.76, SD = 0.44) and human parent images (M = 2.87, SD = 0.52), t17 = −1.076, p = 0.297. Participants Healthy male and female adults with no record of neurological or psychiatric illness and no current medication use volunteered for one of the two studies. Participants in the first experiment, conducted in Wurzburg, were students of the University of Wurzburg and those of the second experiment, conducted in Zurich, were students of the University of Zurich. All participants were native or fluent speakers of Standard German, consistently right-handed (Annett, 1970), and had no previous experience designing or modifying computer-generated characters in, for example, virtual reality-based role-playing games, second life, or virtual reality environments, or experience using such environments (e.g., for psychotherapy, rehabilitation, training, e-commerce, or virtual reality-based research). Written informed consent was obtained before participation according to the guidelines of the Declaration of Helsinki. Each volunteer received 20 Swiss Francs or the equivalent in Euros for participation. The study and all procedures and consent forms were approved by the Ethics Committee of the Universities of Wurzburg and Zurich. Experiment 1: Psychophysiological Recordings and Ratings of Valence and Arousal Participants Of N = 30 participants, three were excluded before data analyses because of excessive impedances during facial EMG and EEG acquisition, leaving N = 27 participants aged between 18 and 36 years (15 female; M = 23 years; SD = 4.02). Materials and Stimuli Twenty linear morph continua were generated, using FantaMorph software (Version 5.3.5, Abrosoft1 ), from 20 different pairs of color images of avatar and natural human faces. Each pair represented the two endpoints of a morph continuum, the continua being used to represent the DHL (for an example of stimuli used in this study, see Figure and Supplementary Figure S1B). Each continuum comprised 13 different morphed images, labeled M0 (avatar endpoint) to M12 (human endpoint), with each morph position representing an equally spaced-point along its respective continuum at increments of 8.33%. All faces were unknown and male, showing full face, frontal view, neutral expression, direct gaze, and no salient features such as facial hair and jewelery. The modeling suite Poser 7 (Smith Micro Software2 ) was used to generate and model in detail the facial geometry and texture (e.g., age, configural cues, skin tone) of the avatar faces to closely match the corresponding human face of the respective continua. The images were then edited in Adobe Photoshop CS3 to mask external features with an elliptic form and black background (96 dpi and 560 × 650 pixels), to ensure final alignment of avatars and human facial features, and to match contrast levels and overall brightness of each pair of parent faces before morphing. The 20 continua (260 stimuli in total) were used to ensure a sufficient number of trials for signal averaging across continua in order to enhance the signal-to-noise ratio for the LPP and EMG measures. The final choice of continua was based on three pilot studies (N = 82). Two of these pilot studies used a two-alternative forced choice classification task to verify the consistency of the category structure of the DHL across the continua. This task required that the participants identify the 1 2 Materials and Procedure All participants were tested individually. Participants were seated in a small, sound-attenuated, dimly lit, shielded cabin, and electrodes for EMG and EEG acquisition were attached. Participants then provided demographic information and completed the State Trait Anxiety Inventory (STAI; Spielberger et al., 1970; German version by Laux et al., 1981). A 1 min resting baseline was performed at the beginning of the experiment to facilitate laboratory adaptation. Each participant received written instructions presented on the screen before commencement of each of three tasks, which were always performed in the same order (in keeping with standardized procedure, e.g., Amrhein et al., 2004). Three tasks were conducted. Each task presented the same 260 stimuli (550 × 650 pixels) at a viewing distance of 62 cm and subtended a visual angle of 11◦ × 14◦ ; this is approximately equivalent to viewing a real face from a normal distance during conversation of 90–100 cm (Hall, 1991; Henderson et al., 2005). The stimuli were always presented individually and in random order, with the constraint that no stimuli from within the same continuum or from corresponding morph positions of the different continua were shown in sequence. In Task 1, participants viewed the stimuli for the duration of each stimulus’ presentation, without any further task requirement. Each trial began with a stimulus that timed out at 750 ms. followed by the inter-trial interval (ITI). The ITI varied randomly (between 4,000 and 5,000 ms), showing a black screen with a white fixation cross. EMG and EEG psychophysiological measures were recorded concomitantly. In Task 2, the stimuli were presented to the participants using a computerized version http://www.abrosoft.com http://www.smithmicro.com Frontiers in Psychology | www.frontiersin.org 4 July 2015 | Volume 6 | Article 981 Cheetham et al. Uncanny hypothesis and effect artifacts (Gratton et al., 1983). Data for the LPP was extracted for stimulus synchronized epochs from 100 ms baseline preceding stimulus onset till 750 ms post-stimulus onset. The data was then baseline corrected (i.e., the 100 ms before stimulus onset), and then averaged for each participant and each of the 13 corresponding morph positions of the 20 continua. The LPPs were determined on the basis of mean amplitude calculated over time windows on the basis of the literature (e.g., Schupp et al., 2000). In particular, LPPs were scored as mean activity between 300 and 750 ms after stimulus onset over the midline electrode sites (CPz, CP1, and CP2; Schupp et al., 2004; Hajcak et al., 2007, 2010; Foti and Hajcak, 2008). All data analyses in this and the following experiment were performed using SPSS version 18.0 (SPSS, Inc., Chicago, IL, USA). of the SAM. This required that participants press an appropriate key to indicate their subjective ratings of valence and arousal for each stimulus; the SAM rating scales range from 1 to 9 (i.e., very positive to very negative for valence and very high to very low for arousal). The button press was followed by the ITI (as in Task 1). A practice pre-test of five trials using stimuli from continua not included in the main test was performed to ensure correct use of the SAM rating scales. In Task 3, a twoalternative forced choice classification task was conducted to verify the location of the morph associated with greatest categorization ambiguity and the profile of avatar and human category decisions for the other morphs; please note that this task is the same as the two-alternative forced choice classification task used in the pilot studies. This task required that participants press an appropriate key to indicate their category judgments. The button press was followed by the ITI (as in Task 1), with time out at 750 ms. A practice pre-test of five trials using stimuli from continua not included in the main test was performed to ensure correct use of the response buttons and comprehension of the category label ‘avatar.’ Results STAI Questionnaire The average score of the STAI state scale was M = 36.07 (SD = 7.96, range = 22–55) and that of the STAI trait scale was M = 36.42 (SD = 10.19, range = 20–60); the STAI scales measure anxiety, ranging from 20 (not at all anxious) to 80 (very anxious). Psychophysiological Data Recording and Reduction Continuous psychophysiological recording was performed in Task 1. EMG acquisition entailed bipolar placement of Ag/AgCl electrodes with surface diameter of 7 mm over the left M. corrugator supercilii (Fridlund and Cacioppo, 1986). Participants were told that skin conductance would be recorded (Dimberg et al., 2000; Weyers et al., 2006, 2009; Likowski et al., 2012). The EMG raw signal was measured with a V-Amp 16 amplifier (Brain Products Inc., Gilching, Germany) and stored with a sampling frequency of 1000 Hz. Raw data were then rectified and filtered offline with a 30 Hz low pass and 500 Hz high pass cut-off filter, a 50 Hz notch filter, and integrated with a 125 ms time constant. The EMG difference scores were computed on the basis of the mean change in activity after stimulus onset from 500 ms baseline before stimulus onset. Trials with EMG activity exceeding 8 µV during the 500 ms baseline and above 30 µV during stimulus presentation were excluded (less than 5%). For statistical analyses, the data of each participant were collapsed over the 20 trials of each of the 13 corresponding morph positions of the 20 continua and averaged over the 100 ms intervals post-stimulus onset (Weyers et al., 2006; van Boxtel, 2010). For the LPP, EEG was recorded using Ag/AgCl electrodes placed at mid-line sites according to the international 10–20 system (i.e., sites FCz, Cz, CPz, Pz, C1, C2, CP1, CP2) at a sampling rate of 1,000 Hz and referenced to Cz during data recording and replaced by the mean of mastoids during off-line data analysis. Electrodes were mounted on an Easycap (EasyCap, Hersching, Germany). Raw data were processed and analyzed using the computer Brain Vision Analyzer software (Version 2.0, Brain Products Inc.). The continuous EEG data were subjected to band-pass between 0.01 and 20 Hz filter off-line. Trials with EEG activity exceeding a transition threshold of 50 µVolt (sample to sample) or amplitude of 300 µV were excluded from further analysis. EEG data was corrected for blinks and eye movement Frontiers in Psychology | www.frontiersin.org Categorization Responses To verify the choice of categorically ambiguous and unambiguous morphs for further analyses, informal inspection of the results of the two-alternative forced choice classification task (Task 3 of Experiment 1), indicates that morph position M6 is associated with greatest ambiguity in avatar-versus-human categorization responses and that the avatar (i.e., M0, M1, M2, M3) and human faces (i.e., M9, M10, M11, M12) show a lower and upper asymptote that nears 95% (see Supplementary Figure S2 in Supplemental Information 2); this profile of category judgments is consistent with the pilot data (see Supplemental Information 1). To characterize this profile more clearly, the mean categorization response data for M6 were compared with the aggregated mean data for the avatar faces and human faces. Greenhouse–Geisser adjustment was applied to correct the degrees of freedom for violation of the sphericity assumption as appropriate in this and subsequent analyses. A one-way repeated measures of analysis of variance (RM-ANOVA) with the factor morph position (three levels: M6, ‘M0, M1, M2, M3’ and ‘M9, M10, M11, M12’) was conducted on the dependent variable categorization response for each participant across continua. The categorization responses were entered in the analysis in terms of percentage of responses categorized as human. This analysis showed a highly significant effect for the expected differences for morph position in categorization responses, F(2,52) = 347.42, p < 0.001, with M6 approaching chance level of 50% in categorization responses (M = 0.44; SD = 0.17), whereas the morphs at M0, M1, M2, and M3 and at M9, M10, M11, and M12 were clearly judged to be exemplars of the avatar (M = 0.04; SD = 0.06) and human face categories (M = 0.9; SD = 0.12), respectively (see Figure 2A). 5 July 2015 | Volume 6 | Article 981 Cheetham et al. Uncanny hypothesis and effect FIGURE 2 | Mean categorization responses and response times in the two-alternative forced choice categorization task. Mean categorization responses are depicted in terms of percentage of ‘human’ responses (A) for morphs representing unambiguous avatar and human faces and the most ambiguous face along continua representing the DHL. The depiction of corresponding mean response times for categorization responses (B) show longer response latencies for ambiguous faces and the shortest response latencies for avatar faces. The error bars indicate standard errors (N = 27). Categorization Response Times (RTs) For lpp, there was a significant effect of morph position along the DHL, F(1.31,34.09) = 5.62, p = 0.016. Pre-planned contrasts showed a significant difference between the avatar (M = 10.92; SD = 3.54) and human faces (M = 9.31; SD = 3.5), F(1,26) = 36.31, p < 0.001, such that the measure for LPP was greater for the avatar than for the human category (see Figures 3A and 4). The pre-planned contrasts showed no significant difference between M6 and the avatar or the human faces, respectively. These data indicate that the LPP values increased across the three stimulus conditions (i.e., avatar faces, M6 and human faces) with increasing morph distance from the human end of the continua. For EMG and the corrugator supercilii activity (see Figure 3B), there was no significant effect of morph position, F(1.38,35.83) = 0.66, p = 0.47. There was a significant effect of morph position for the SAM arousal ratings, F(1.51,39.38) = 3.78, p = 0.043. Preplanned contrasts showed a significant difference between the avatar morphs (M = 3.6; SD = 1.54) and M6 (M = 3.38; SD = 1.49), [F(1,26) = 4.43, p = 0.045] and between the avatar and the human morphs (M = 3.34; SD = 1.58), [F(1,26) = 4.45, p = 0.045], such that arousal ratings increased across the three stimulus conditions with increasing distance from the human end of the continua (see Figure 3C). There was no significant difference between arousal ratings for M6 and human faces. For the SAM valence ratings, there was a highly significant effect of morph position along the DHL, F(1.29,33.65) = 56.69, p < 0.001. Pre-planned contrasts showed a significant difference between avatar faces (M = 5.68; SD = 0.62) and M6 (M = 4.91; SD = 0.51), [F(1,26) = 59.75, p < 0.001], between M6 and the human faces (M = 4.55; SD = 0.68), [F(1,26) = 24.63, p < 0.001], and between avatar and human faces [F(1,26) = 36.31, p < 0.001]. These data show that the valence ratings increased negatively The longest response latency might be expected to correspond with greatest categorization ambiguity along the DHL (Cheetham et al., 2011, 2013). To confirm this for the present data (though RT is not relevant for the Tasks 1 and 2 in Experiment 1), the same RM-ANOVA as in the preceding was applied using categorization response times (RTs; in ms) rather than categorization response as the dependent variable. Given the reported asymmetries in processing avatar and human faces along the DHL (Cheetham et al., 2011, 2013), this and the following analyses included pre-planned contrasts to compare measures for M6 and the human and avatar faces. This analysis showed a significant effect for morph position along the DHL, F(1.11,29.06) = 17.13, p < 0.001. The pre-planned contrasts showed a significant difference between RT at M6 (M = 1689; SD = 1091) compared with the mean average for avatar and human morphs (M = 1097; SD = 551), F(1,26) = 20.41, p < 0.001, indicating that RT is significantly longer at M6 than for other morphs. Consistent with previous RT data (Cheetham et al., 2011, 2013; Cheetham and Jancke, 2013), pre-planned contrasts showed a significant difference in RT between the avatar (M = 924; SD = 276) and human morphs (M = 1258; SD = 534), F(1,26) = 22.58, p < 0.001, such that the mean latency of categorization responses was longer for human faces (see Figure 2B). Affective Experience and Categorization Ambiguity Based on the preceding, we compared the lpp, emg, sam for valence and sam for arousal data at m6 with the data for the avatar and human category faces. Separate one-way rm-anovas with the factor morph position (three levels: m6, ‘m0, m1, m2, m3’ and ‘m9, m10, m11, m12’) were conducted for each of the dependent variables lpp, emg, sam ratings for valence, and sam ratings for arousal of each participant across the 20 continua. Frontiers in Psychology | www.frontiersin.org 6 July 2015 | Volume 6 | Article 981 Cheetham et al. Uncanny hypothesis and effect FIGURE 3 | Effect of highly unambiguously avatar and human and highly ambiguously categorized faces on arousal and valence. Overall, the figures illustrate a general increase in LPP magnitude indicating arousal (A), in self-rated arousal (C) and in self-rated negative valence (D) with decreasing human likeness from the human to the avatar faces. Differences in EMG magnitude indicating negative valence (B) are not significant. The error bars indicate standard errors (N = 27). across the three stimulus conditions with increasing morph distance from the human end of the continua (see Figure 3D). Please note that the levels ‘M0, M1, M2, M3’ and ‘M9, M10, M11, M12’ were selected for the preceding analyses to represent the avatar and human categories. But performing the same separate one-way RM-ANOVAs using just M0, M6, and M12 as the morph factor levels produced the identical pattern of results for LPP, EMG, SAM ratings for valence, and SAM ratings for arousal. possible after stimulus onset. This task permitted the analysis of RTs for familiarity judgements. The rating scale ranged from very strange (1) to very familiar (5). A practice pre-test of five trials was applied, as described for the tasks of Experiment 1. Please note that the pilot study to determine facial attractiveness of continua endpoints, described in Section “Materials and Stimuli,” was the same as used for self-ratings of familiarity, except that a 5-point bipolar Likert rating scale ranging from very unattractive (1) to very attractive (5) was used. Experiment 2: Ratings of familiarity Results Participants and Procedure Familiarity Ratings A sample of N = 30 participants aged between 20 and 30 years (15 female; M = 25.64 years; SD = 2.88) were examined. The laboratory, stimulus conditions, task requirements, and instructions in this study were the same as described for Task 2 (i.e., self-ratings of valence and arousal) of Experiment 1, except that participants were required to view and rate feelings of familiarity in response to stimuli on a 5-point Likert scale by pressing the appropriate response key as quickly and accurately as A one-way RM-ANOVA with the factor morph position (three levels: M6, ‘M0, M1, M2, M3’ and ‘M9, M10, M11, M12’) and the dependent variables familiarity rating of each participant across the 20 continua revealed a highly significant effect of morph position, F(1.27,36.85) = 109.03, p = <0.001. Pre-planned contrasts showed a significant difference between the avatar morphs (M = 1.9; SD = 0.72) and M6 (M = 3.01; SD = 0.54), [F(1,29) = 134.42, p = <0.001] and between M6 and the human Frontiers in Psychology | www.frontiersin.org 7 July 2015 | Volume 6 | Article 981 Cheetham et al. Uncanny hypothesis and effect Discussion The main prediction of the UVH is that observation of highly humanlike characters or objects that are difficult to distinguish from the human counterpart will elicit negative affect, whereas the affective experience of distinctly non-human and human characters or objects will be more positive in comparison. To test this, measures of valence and arousal (i.e., the primary orthogonal dimensions of affective experience) were used to compare the impact on affective state of categorically ambiguous faces with that of categorically unambiguous avatar and human faces; the physical morph distance of the avatar and human faces from the ambiguous faces was controlled for. To reflect Mori’s idea, we assumed that any evidence in support of an uncanny-like effect (i.e., enhanced negative affective experience for ambiguous faces) during passive viewing and during explicit affect evaluation would most likely be revealed by comparing the categorically most ambiguous stimuli with the most unambiguous stimuli. But the data from the LPP, EMG, and SAM-based measures of arousal and valence converge in showing no support for the notion that category ambiguity along the DHL is specifically associated with enhanced experience of negative affect. On the contrary, the LPP and SAM-based measures indicate a general increase in arousal and negative valence across the stimulus conditions (i.e., the human category, ambiguous category, avatar category) with increasing morph distance from the human end of the continua. The ad hoc familiarity ratings delivered the same picture, indicating that feelings of strangeness are not specifically associated with categorically ambiguous faces and generally increase with increasing morph distance from the human end of the continua. These findings are consistent with the profile of subjective evaluations reported in other studies that have used comparable, well-controlled morph continua and ad hoc measures of shinwakan, such as pleasantness (e.g., Looser and Wheatley, 2010). This profile is characterized by a general asymmetry in affective experience, with increasingly negative evaluations of morphs with decreasing human likeness. But asymmetry along the DHL is not specific to affective processing. Asymmetries have also been reported in tasks of perceptual and category processing in other uncanny-related studies (Cheetham et al., 2011, 2013, 2014). These tasks have revealed greater decision certainty and shorter RT latencies in categorization judgments for avatar compared with human faces, extraction of different perceptual details from avatar compared with human faces during perceptual decision making, differential sensitivity of affect-related brain structures (e.g., amygdala, insula) to avatar compared with human faces during passive viewing, and enhanced discrimination sensitivity to perceptual differences in visual information between faces within the avatar compared with those within the human category. The data of the present study are therefore worth considering in the context of such asymmetries in perceptual and category processing. The RT data in the categorization task (i.e., Task 3 of Experiment 1) show that explicit categorization judgments FIGURE 4 | Late positive potential (LPP) waveforms and scalp topographical distribution. The figure illustrates the LPP waveforms (A) and the scalp topographical distribution of the LPP (B) for avatar faces, ambiguous faces and human faces. morphs (M = 3.68; SD = 0.54), F(1,29) = 48.58, p = <0.001, such that familiarity ratings increased negatively across the three stimulus conditions with increasing distance from the human end of the continua (see Figure 5A). RT of Familiarity Ratings The same analysis, using instead RT for familiarity ratings also showed a highly significant effect of morph position, F(1.27,36.85) = 12.71, p = <0.001 (see Figure 5B). Reflecting the pattern of RT for categorization in Task 3 of Experiment 1, pre-planned contrasts showed a significant difference between RT at M6 (M = 1682; SD = 527) compared with the mean average for avatar and human morphs (M = 1482; SD = 376), F(1,26) = 20.41, p < 0.001, indicating that RT for familiarity judgments is significantly longer at M6 than for categorically unambiguous morphs. Pre-planned contrasts showed also that RT for familiarity ratings of avatars (M = 1400; SD = 453) was significantly faster than for human faces (M = 1627; SD = 437), F(1,29) = 11.9, p = 0.002. Frontiers in Psychology | www.frontiersin.org 8 July 2015 | Volume 6 | Article 981 Cheetham et al. Uncanny hypothesis and effect and human faces. The error bars indicate standard errors (N = 30). (B) Shows examples of morphs drawn from a continuum of the kind used to represent the DHL. The morph labeled M0 corresponds with the avatar endpoint of the continuum, M6 with greatest category ambiguity, and M12 with the human endpoint of the continuum. (C) Shows examples of stimuli used in this study. FIGURE 5 | Self-reported feelings of familiarity. (A) Illustrates a general increase in familiarity ratings with increasing human likeness from the avatar to the human faces (A). Reflecting the pattern of response times for categorization response times in the two-alternative forced choice categorization task, response latencies for ambiguous faces are longer compared with the avatar of category did occur, a similar RT facilitation effect might be expected for our affective evaluations of avatar faces (please note that for experimental reasons, RT was only collected for familiarity judgments). This suggestion assumes that processing of category membership contributes to or influences in some way the processing of familiarity (for categorization effects and the evaluation of attractiveness, see Halberstadt and Winkielman, 2014). In fact, the data show that the latency of familiarity judgments is shorter for avatar than for human faces. The data show also that the latency of familiarity judgments is longest for the categorically most ambiguous faces. Familiarity judgments thus appear to be influenced by or interact with the processing and cognitive representation of the category structure of the continua (cf., e.g., Heekeren et al., 2008). Similar effects might apply for the psychophysiological and for the SAM-based measures of valence and arousal. The use of a different task design that allows clear interpretation of RT data of the SAMbased measures of valence and arousal might be used to examine this. The preceding considerations hint at the possibility that there is a relationship of some kind between cognitive processing efficiency (as indicated in the preceding by RT), perceptual and category processing of the DHL, and our measures of affective experience of DHL stimuli. Further analysis of the familiarity ratings supports the idea of a relationship, revealing a highly significant correlation for avatars only, such that shorter RT for category judgments of avatars is associated for avatar faces are faster than those for human faces. One interpretation of this finding is that different perceptual features are used for processing category information in novel avatar compared with “everyday” human faces, as indicated for implicit and explicit processing of perceptual and category information along the DHL in other studies (Cheetham et al., 2011, 2013, 2014). It has been suggested that this difference might relate to the use of novel and thus salient perceptual information as a readily identifiable feature of avatar faces (e.g., novel color, smoothed skin texture, or feature shape) to facilitate category processing (Cheetham et al., 2013). Assuming that novel information is easier to extract from the avatar faces compared with the corresponding category information from the human faces and that this information is preferentially used as diagnostic of avatar category membership, one might expect an RT facilitation effect such that RT latencies for category judgments of novel avatar faces are shorter compared with those of human faces (see Levin and Angelone, 2001). The present data are consistent with this idea (for an alternative explanation of RT facilitation, see Valentine, 1991). While there was no requirement to categorize our stimuli during the other tasks (i.e., during passive observation, explicit evaluation of affect, and familiarity judgments), it is likely that participants did engage in implicit processing of the categories (for evidence of this during passive observation, see Cheetham et al., 2011; see also Castelli et al., 2004). If implicit processing Frontiers in Psychology | www.frontiersin.org 9 July 2015 | Volume 6 | Article 981 Cheetham et al. Uncanny hypothesis and effect is entirely contrary to the effect predicted on the basis of the UVH. The reported increase in negative valence and arousal for avatar compared with the human faces in the present study might simply relate to an innate predisposition to treat the unfamiliar with caution (Zajonc, 1998). The strength of caution diminishes as further exposure reveals that the unfamiliar is nonthreatening (Lee, 2001). Correspondingly, the relatively more positive evaluations of valence for human faces might simply reflect the impact of repeated exposure to human category examplars. Previous social interaction and the often more positive affective tone of interaction with a particular in-group is thought to lead to automatic activation of more positive evaluations (Reis and Gable, 2003; Claypool et al., 2007; GarciaMarques et al., 2010). This mere-exposure effect (Zajonc, 1968; Monahan et al., 2000; Zajonc, 2001) might underpin the general increase in pleasantness and liking ratings with increasing human likeness of faces in other studies (e.g., Looser and Wheatley, 2010; Cheetham et al., 2014; see Experiment 1 in Seyama and Nagayama, 2007), such that more humanlike faces (or their human-specifying perceptual features) are evaluated as more likeable (see, Moreland and Zajonc, 1982). A recent study investigated the idea that the frequency of exposure to the faces of fictive beasts modulates affective ratings of eeriness (Burleigh and Schoenherr, 2015), but the authors report only nearly significant effects. Given that these fictive faces were not manipulated in terms of human likeness, the potential impact of mere-exposure on affective ratings of highly humanlike faces is open to further consideration. One possible consideration is that the mere-exposure effect is mediated by the history of normal social interaction and a tendency to individuate in-group but not out-group members (Ostrom et al., 1993). This differential processing bias might also apply when processing human and humanlike faces (see Cheetham et al., 2013). This bias means that human participants preferentially code other members of the human in-group (i.e., our human stimuli) by directing cognitive processing resources toward more in-depth processing of facial information to enable individuation (i.e., processing at the exemplar level). In contrast, the processing of out-group members (i.e., our highly humanlike avatar faces) might be biased toward facial information that enhances detection of faces at the category level; for this kind of out-group bias by other names, see the otherrace hypothesis (Levin, 2000), differential processing hypothesis (Ostrom et al., 1993), and the other-race effect (Rhodes et al., 2009). More in-depth processing for individuation would be consistent with the longer RT latencies for categorization of our human category faces in Task 3 of Experiment 1, since longer latency suggests more time-consuming processing of finer perceptual details (Schyns and Murphy, 1994; Lamberts, 1998; Johansen and Palmeri, 2002). Assuming that longer RT of familiarity ratings for human faces also reflects more in-depth processing, the allocation of more attentional resources needed for this might be sufficient to strengthen any effects of mereexposure on positive evaluations of faces (Huang and Hsieh, 2013). This explanation is consistent with the present data and it could be investigated further in relation to categorization with more negative familiarity ratings (i.e., greater strangeness), r = 0.575, p > 0.001. This finding is not consistent with the recent inhibitory-devaluation hypothesis that has been presented as a potential explanation for the uncanny valley (Ferrey et al., 2015). Ferrey et al.’s (2015) hypothesis posits that a stimulus that is subject to competing interpretations, such as when membership of a stimulus to one or other potential category is ambiguous, will be evaluated more negatively (for further details regarding the role of inhibitory cognition in this). Based on the use of RT to indicate decision difficulty due to competing interpretations of categorically ambiguous stimuli (see also Yamada et al., 2013), our data reveal no significant relationship between RT and familiarity ratings for categorically ambiguous faces and that the only significant relationship found (i.e., for avatar faces) shows longer RT for more positive ratings. The data of both experiments in Burleigh and Schoenherr’s (2015) recent study, based on ratings of eeriness, also lend no support for the inhibitory-devaluation hypothesis. An alternative account of the uncanny effect that has attracted attention in uncanny research considers the influence of processing fluency on affective experience. According to the Hedonic Fluency Model (Winkielman et al., 2003), negative evaluations of novel or unfamiliar stimuli relate to subjective difficulty extracting diagnostic information for quick and efficient processing (see also Bradley et al., 1993; Bornstein and D’Agostino, 1994; Mendes et al., 2007). This proposal ties in well with the idea that negatively valenced experience along the DHL might be associated with category ambiguity. Yamada et al. (2013) follow the Hedonic Fluency Model and suggest on the basis of their data that lower processing fluency, as indicated in their study by categorization decision difficulty (i.e., longer RT) for categorically ambiguous stimuli of the DHL, is associated with enhanced negative judgments of likeability. In contrast, our data indicate that any change in arousal, valence and familiarity is not modulated by effects of category ambiguity and, specifically in relation to the correlative result between RT and the faces of the avatar category, do not favor the hedonic fluency account. It might be argued that the correlative relationship between RT and familiarity ratings for avatars fits more closely with a different model of processing fluency, the Fluency Amplification Model (Albrecht and Carbon, 2014). Albrecht and Carbon show that stimuli with a comparatively neutral or negative valence at the outset are not liked any more under conditions of higher processing fluency than they are under conditions of lower processing fluency. They show also that higher processing fluency of negative stimuli can actually enhance negative evaluation. Our data are consistent with the possibility that higher processing fluency of avatar faces (i.e., shorter RT latencies) and enhanced negative evaluation might be related in this way. This possibility is reinforced by the data of a recent study showing that higher processing fluency, indicated by reduced difficulty in perceptual discrimination between highly similar faces along the DHL, correlates with negative affect evaluations of familiarity (i.e., enhanced feelings of strangeness; Cheetham et al., 2014); please note that this finding Frontiers in Psychology | www.frontiersin.org 10 July 2015 | Volume 6 | Article 981 Cheetham et al. Uncanny hypothesis and effect reflect this general decrease and not produce an uncanny-like effect. But further investigations could examine this possibility by using more trials per morph level. It should be noted also that while the LPP is modulated by arousing (negatively and positively valenced) stimuli (e.g., Schupp et al., 2000; Leite et al., 2012; Weinberg et al., 2012), valence can also modulate the LPP (e.g., Cuthbert et al., 2000; Delplanque et al., 2006). The valence effect is less consistently reported (Olofsson et al., 2008), but we cannot exclude the possibility that effects of both arousal and valence are reflected in the LPP data. This would not change the findings of the present study, as arousal and valence are primary dimensions of affective experience. The use of psychophysiological indices of affect introduces additional sources of data to the investigation of Mori’s ideas. Together with the SAM-based ratings, this approach delivers a more complete picture of the underlying components of affective experience. In the present study, this picture includes direct and objective measures of psychophysiological reactions to the DHL stimuli and indirect and subjective measures based on the cognitive evaluation of the stimuli and of the consciously detected psychophysiological reactions that these evoke. The appeal of psychophysiological indices is that their measurement is conducted continuously and in real time, meaning that physiological events can be detected as they unfold over time at a temporal resolution on a millisecond scale. For example, the LPP develops at around 300–400 ms after stimulus onset, peaks at around 700 ms, and lasts for up to 6 s in total (Cuthbert et al., 2000). Similarly, EMG can provide effective measurement of minuscule and rapid changes, including changes that escape detection by the naked eye (e.g., Cacioppo et al., 1986; Dimberg, 1990; Weyers et al., 2006; Gomez et al., 2009; Mauss and Robinson, 2009; van Boxtel, 2010; Likowski et al., 2011; Wu et al., 2012). In view of the poor construct definition of the affective dimension described in the UVH, that some individuals find it difficult to conceptualize and quantify their emotional experiences (Mauss and Robinson, 2009), and that (ad hoc) self-reports might not well capture the constructs that they are intended to measure (Ho et al., 2008), further use of psychophysiological measures might contribute to a clearer characterization of the relationship between stimuli defined along the DHL and affect. To characterize this relationship and to enable comparison between studies, the way in which the DHL is represented is an important consideration. One approach to its representation is to use morph continua (e.g., Seyama and Nagayama, 2007; for an alternative approach, see, e.g., Ho et al., 2008; Tinwell, 2009). The use of morphing permits close examination of the relationship between affect and experimentally controlled fine-grained differences in humanlike appearance. The general increase in positive affect toward the human end of the DHL indicated by our LLP, SAM arousal, and SAM valence (and by our familiarity) measures is reflected in the data of other studies based on subjective ratings of affect and on comparable nonhuman– human morph continua (e.g., Experiment 1 in Seyama and Nagayama, 2007; Looser and Wheatley, 2010; Cheetham et al., 2014). But there are findings inconsistent with the present data. The most similar study is that of Yamada et al. (2013). performance. It is worth noting, however, that Cheetham et al. (2014) did already test the differential processing bias as a potential explanation for their finding of an asymmetry along the DHL in perceptual discrimination, that study showing enhanced perceptual discrimination of avatar faces compared with human faces. The data in that study did not support this explanation, and, in terms of perceptual discrimination, lend little support to Schoenherr and Burleigh’s (2015) suggestion that an out-group bias might also underpin certain social-cultural phenomena that resemble elements of the uncanny valley idea. We applied a two-dimensional approach to examining affective experience by placing the focus in the main experiment on arousal and valence. The data show a consistent pattern in the relationship between lesser degrees of human likeness and greater arousal (i.e., in the LPP and self-rating SAM measures) and more negative valence (i.e., in the self-rating SAM measure). The combination of negative valence and increased arousal is understood as indicating negative affective experience (Lang et al., 2005). But the EMG measure of corrugator supercilii activity showed no effects along the DHL. A straightforward interpretation of this would be that human likeness along the DHL has no differential impact on the valence of actual affective state during passive viewing. But in keeping with Larsen et al. (2003), who report a strong relationship between corrugator supercilii activity and self-reported valence ratings, further analysis of the data of each participant across continua also shows a strong positive relationship between increasing corrugator activity and more negative valence ratings (r = 0.474, p = 0.006). However, this effect is specific for the human category, suggesting that the valence of actual feeling state (as indexed by the EMG measure) and the cognitive evaluation of feeling state (as indexed by SAM measure) strongly converge for human faces only. This convergence might reflect a close coupling of the representation and integration of affective and cognitive processing of actual feeling state and of the cognitive appraisal of that state in relation to external input (Schwarz and Clore, 1983, 2006) that might be acquired through repeated exposure and social interactive experience with human others. This humanspecific effect might relate to the processing of facial mimicry acquired in normal social interaction with human others (Weyers et al., 2006). The comparatively weak effect for non-human faces might thus reflect an attenuated responsiveness to the neutral expression of non-human faces. The data show a significantly greater (i.e., more positivegoing) LPP for the avatar compared with the human category faces. It is possible that a larger number of trials (i.e., use of more continua) might have increased the chance of also finding a significant difference between these two face categories and the ambiguous faces. Huffmeijer et al. (2014) recommends 30 trials to capture the LPP, whereas Moran et al. (2013) demonstrate that the LPP is stable and can be quantified with as few as 12 trials. Based on previous studies (Cuthbert et al., 2000; Yen et al., 2010; Herbert et al., 2013; Moran et al., 2013), we considered 20 continua to be adequate to test Mori’s ideas. Considered in the context of the general decrease in LPP amplitude with increasing human likeness of the morphs, it is likely that any such difference between the ambiguous and the unambiguous faces would still Frontiers in Psychology | www.frontiersin.org 11 July 2015 | Volume 6 | Article 981 Cheetham et al. Uncanny hypothesis and effect The present study focused on affective experience in terms of the dimensions arousal and valence. Affective experience can be conceptualized in other ways (see e.g., Davidson, 1993; Panksepp, 1998; Lindquist et al., 2012), and examination of arousal and valence is not new in emotion research (e.g., Lane et al., 1999; Robinson and Compton, 2006; Lewis et al., 2007; Demanet et al., 2011). But this two-dimensional approach is different than that taken in uncanny research to date (e.g., Ho et al., 2008; Tinwell, 2009; Ho and MacDorman, 2010). For example, Ho et al. (2008) focus on defining specific emotions, such as fear, with which to characterize uncanny experience. But it is worth noting that Ho et al. (2008) see parallels between their own findings (using robotic stimuli), arousal and valence, and Russell’s circumplex model of affect (Russell, 1980). This model understands affective states, such as fear, as arising from neurophysiological systems that relate to arousal and valence (for a detailed review, see Posner et al., 2005). It is conceivable that measures of arousal and valence might explain a significant amount of the variance in and provide further insight into the affective constructs (e.g., likeability, feelings of familiarity, fear, disgust) typically used to investigate the uncanny effect (for overviews, see Ferguson and Bargh, 2003; Posner et al., 2005; Cunningham and Zelazo, 2007; Schwarz, 2007). We note, however, that psychophysiological measures do not replace measures of self-reported feelings, because self-reports and measures of psychophysiological reactivity and behavior are all relevant to the description of an emotional response (Mandler et al., 1961; Lang, 1989). Whether the present findings might generalize to fine-grained manipulations of the DHL based on computer-generated stimuli using different software to generate avatars (with manipulations of different perceptual information), female face stimuli, emotionally expressive faces, and dynamic stimuli (see Chaminade et al., 2007) is open to further investigation. They examined the relation between explicit ratings of likeability and category ambiguity along a morph continuum generated from the face of the cartoon character Charlie Brown and a human face. While they reported negative affect specifically in relation to category ambiguity, they indicate also that morphing disparities in the alignment of facial features may have influenced subjective ratings. Inspection of their stimuli suggests that these disparities particularly affected the categorically most ambiguous morphs by creating the appearance of a facial scar across the forehead of the morphed faces. This kind of morphing artifact is likely to have had a systematic effect on subjective ratings as it is related to the morph distance from the continua endpoints (see Cheetham and Jancke, 2013). MacDorman and Ishiguro (2006) also used non-human and human morphs to represent the DHL, reporting an uncanny valley-like negative peak in affective ratings. But their use of more than one juxtaposed continuum to represent the DHL combined with non-equivalent increments of physical change between the morphs of the DHL (see also Hanson et al., 2005) appears to have created the stimulus conditions needed to generate an uncanny-like effect in the profile of subjective responses. It should be noted that the experimental manipulation of facial features along morph continua for the explicit purpose of evoking uncanny-like effects has been applied in other experiments (see Experiments 2 and 3 in Seyama and Nagayama, 2007). Other studies have used experimentally controlled morph continua and examined affect along similar dimensions of facial human likeness. However, these have used computer-generated rather than natural human faces to represent the human end of the human likeness dimension (MacDorman et al., 2009a). Considered in terms of the UVH, the human endpoints in these studies are in effect exemplars of the non-human category. Use of computer-generated faces to represent the human faces is not unusual in face research (e.g., Todorov et al., 2009). But there are, as discussed in the preceding, differences in perceptual and category processing between natural human and computer-generated human-like faces. Importantly, perceptual and category processing of human category and similar computer-generated, non-human category faces can correlate differently with measures of affect, as shown for perceptual discrimination (Cheetham et al., 2014) and in the present study. In terms of the UVH, this makes comparison between such studies and those that use natural human faces difficult. Burleigh et al. (2013) and Burleigh and Schoenherr (2015) have also investigated the UVH using experimentally controlled morph continua, but their face morphs were not manipulated in terms of human likeness. Acknowledgments This work was supported by the European Union FET Integrated Project PRESENCCIA (Contract number 27731) and by the German Research Foundation (GRK 1253/1 scholarship to LW, FOR 605-PA 566/9-1, and SFB-TRR 58 project B01). Supplementary Material The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpsyg. 2015.00981 References to startle reflex and skin conductance response? Int. J. Psychophysiol. 54, 231–240. doi: 10.1016/j.ijpsycho.2004.05.009 Annett, M. (1970). A classification of hand preference by association analysis. Br. J. 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(2013). “More human than human: does the uncanny curve really matter?” in Proceedings of the HRI2013 Workshop on Design of Humanlikeness in HRI from uncanny valley to minimal design. Tokyo. 7–13. 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. Copyright © 2015 Cheetham, Wu, Pauli and Jancke. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 15 July 2015 | Volume 6 | Article 981 

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sensors Article Monitoring Off-Shore Fishing in the Northern Indian Ocean Based on Satellite Automatic Identification System and Remote Sensing Data Jie Li 1,2,3 , Qianguo Xing 1,2,3, * , Xuerong Li 1 , Maham Arif 1,2,3 and Jinghu Li 1,2,3 1 2 3 * Citation: Li, J.; Xing, Q.; Li, X.; Arif, M.; Li, J. Monitoring Off-Shore Fishing in the Northern Indian Ocean Based on Satellite Automatic Identification System and Remote Sensing Data. Sensors 2024, 24, 781. https://doi.org/10.3390/s24030781 Academic Editors: Javier Fernández Calleja, Susana del Carmen Fernández Menedez and José González-Piqueras Received: 21 December 2023 CAS Key Laboratory of Coastal Environmental Processes and Ecological Remediation, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China; lijie185@mails.ucas.ac.cn (J.L.); xrli@yic.ac.cn (X.L.); jinghuli@yic.ac.cn (J.L.) Shandong Key Laboratory of Coastal Environmental Processes, Yantai 264003, China University of Chinese Academy of Sciences, Beijing 100049, China Correspondence: qgxing@yic.ac.cn Abstract: Satellite-derived Sea Surface Temperature (SST) and sea-surface Chlorophyll a concentration (Chl-a), along with Automatic Identification System (AIS) data of fishing vessels, were used in the examination of the correlation between fishing operations and oceanographic factors within the northern Indian Ocean from March 2020 to February 2023. Frequency analysis and the empirical cumulative distribution function (ECDF) were used to calculate the optimum ranges of two oceanographic factors for fishing operations. The results revealed a substantial influence of the northeast and southwest monsoons significantly impacting fishing operations in the northern Indian Ocean, with extensive and active operations during the period from October to March and a notable reduction from April to September. Spatially, fishing vessels were mainly concentrated between 20◦ N and 6◦ S, extending from west of 90◦ E to the eastern coast of Africa. Observable seasonal variations in the distribution of fishing vessels were observed in the central and southeastern Arabian Sea, along with its adjacent high sea of the Indian Ocean. Concerning the marine environment, it was observed that during the northeast monsoon, the suitable SST contributed to high CPUEs in fishing operation areas. Fishing vessels were widely distributed in the areas with both mid-range and low-range Chl-a concentrations, with a small part distributed in high-concentration areas. Moreover, the monthly numbers of fishing vessels showed seasonal fluctuations between March 2020 and February 2023, displaying a periodic pattern with an overall increasing trend. The total number of fishing vessels decreased due to the impact of the COVID-19 pandemic in 2020, but this was followed by a gradual recovery in the subsequent two years. For fishing operations in the northern Indian Ocean, the optimum ranges for SST and Chl-a concentration were 27.96 to 29.47 ◦ C and 0.03 to 1.81 mg/m3 , respectively. The preliminary findings of this study revealed the spatial–temporal distribution characteristics of fishing vessels in the northern Indian Ocean and the suitable ranges of SST and Chl-a concentration for fishing operations. These results can serve as theoretical references for the production and resource management of off-shore fishing operations in the northern Indian Ocean. Revised: 17 January 2024 Accepted: 23 January 2024 Published: 25 January 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Keywords: automatic identification system (AIS); fishing vessels; spatial–temporal distribution; northern Indian Ocean; sea surface temperature (SST); chlorophyll a concentration (Chl-a) 1. Introduction The ocean serves as the vital blue granary, offering humanity abundant animal proteins and providing economic and employment-related social benefits [1]. Marine fisheries play a crucial role in the marine economy; understanding the changes in fishery resources could promote the economic growth of coastal countries. Currently, with the continuous increase in fishing vessels and progressive fishing technology, understanding the distribution ranges, fishing intensity and other information about fishing operations can indirectly reveal the Sensors 2024, 24, 781. https://doi.org/10.3390/s24030781 https://www.mdpi.com/journal/sensors Sensors 2024, 24, 781 2 of 19 changes in fishery resources. This information could provide necessary decision-making support for marine spatial planning and ecological conservation. The Indian Ocean fishing ground is one of the world’s distant water operational fishing grounds [2,3], holding vast potential for fishery resources. The tropical continental shelf waters of the Indian Ocean are the most important fishing areas, hosting a variety of fish species with high reproductive rates, short reproductive cycles, and significant variations in catch quantities [4–6]. For instance, the Arabian Sea is home to abundant species such as the Scombridae and Clupeidae [7], while the coastal areas of the Bay of Bengal host a large population of catfish species [8]. According to the latest statistics from the Food and Agriculture Organization of the United Nations (FAO), the catch in the Indian Ocean reached 1.22 × 107 tons in 2020 [9]. In this context, it is necessary to understand the distributions of fishery resources in the Indian Ocean. Studying the spatial–temporal distributions and variations of Indian Ocean fishing vessels through vessel monitoring could indirectly reach this goal. Fishing logbooks and Vessel Monitoring System (VMS) are traditional methods for monitoring fishing vessels, providing effective ways to analyze the spatial–temporal distribution characteristics of fishing vessels by recording information such as navigation time, locations, and status. However, both of these methods have their limitations: fishing logbooks need to be brought back only after the vessels return to ports, and these logbooks often suffer from issues such as incomplete information and irregular entries because of the long periods of off-shore operations, leading to shortcomings in the accuracy and effectiveness [10]; VMS data also have limitations, including a low time resolution and restricted access [11,12]. Consequently, the accuracy and timeliness of the two kinds of data are insufficient, and there are certain defects in the analysis of the spatial–temporal distributions and change characteristics of fishing vessels by using these data. The collection and use of real-time fishing data have provided robust support in recent years by technological advancements such as big data. Automatic Identification System (AIS) is extensively employed for real-time monitoring and identification of fishing vessels. It transmits information such as latitude, longitude, heading, and speed, thereby serving as a valuable data source for the monitoring and management of marine fishing operations. The development of satellite AIS further expands AIS applications in the prediction and analysis of the distribution of fishery resources and the location of central fishing grounds [13]. Ocean fishing vessels transmit data to ground stations through satellites, facilitating rapid information feedback with high data accuracy. This mechanism serves to effectively compensate for limitations in fishing logbooks and VMS data [14,15], enabling a better way to complete the characteristics mining of fishing operations. AIS is a digital device and navigation system that utilizes network, communication, and electronic information display technology. It has been widely applied in the maritime field. With the significantly increasing installation rate of AIS devices on fishing vessels, research on fishery-related applications based on AIS data has been widely conducted. Researchers worldwide have achieved a series of achievements in fishing behavior identifications [16–18], fishing operation areas extractions [19], fishing intensity measurements [20,21], and ecological pressure assessment from fishing operations [22,23]. Therefore, AIS data could provide an effective way to study the spatial–temporal distribution and variation characteristics of fishing vessels in the Indian Ocean. Compared with the southern waters, there are many coastal countries in the northern Indian Ocean, resulting in more complex traffic conditions. In this regard, this study leverages AIS data spanning from March 2020 to February 2023 to investigate the spatial– temporal distribution and variation characteristics of fishing vessels in the northern Indian Ocean, indirectly revealing the spatial–temporal distribution of fishery resources and providing information and technical support for marine ecological protection and marine spatial planning and management in the Indian Ocean. Sensors 2024, 24, x FOR PEER REVIEW Sensors 2024, 24, 781 3 of 19 2.1. Study Area and Data 2. Data andstudy Methods The area covers the northern Indian Ocean from 32° E to 100° E and 31° 2.1. Study Area and S, including the Data Red Sea, the Gulf of Aden, the Persian Gulf, the Gulf of Oman, the ◦ ◦ ◦ area covers Sea, the northern Ocean and fromthe 32 Andaman E to 100 E and N to Sea,The thestudy Lakshadweep the BayIndian of Bengal, Sea.31The coastal c 6◦ S, including the Red Sea, the Gulf of Aden, the Persian Gulf, the Gulf of Oman, the and their corresponding Exclusive Economic Zones (EEZs) are illustrated in Figur Arabian Sea, the Lakshadweep Sea, the Bay of Bengal, and the Andaman Sea. The coastal area is influenced by the IndianExclusive Ocean monsoon, featuring thearesouthwest countries and their corresponding Economic Zones (EEZs) illustrated monsoon in ing from June to September and the northeast monsoon from December to March of Figure 1. This area is influenced by the Indian Ocean monsoon, featuring the southwest monsoon prevailing to September and the northeast monsoon from December year. April to Mayfrom andJune October to November are transition periods for the monso to March of the next year. April to May and October to November are transition periods The monsoon transition induces alterations in ocean currents, subsequently im for the monsoons [24]. The monsoon transition induces alterations in ocean currents, upwelling, seawater temperature, salinity, dissolved oxygen, andoxygen, the distribution o subsequently impacting upwelling, seawater temperature, salinity, dissolved and tondistribution and otherofmarine organisms [25]. Consequently, these changes impact the spati the plankton and other marine organisms [25]. Consequently, these changes impact the spatial distribution of fishery resources. bution of fishery resources. Figure1. 1. The northern Indian Figure The northern Indian Ocean.Ocean. Data regarding the locations of fishing vessels were acquired from the MarineTraffic Data regarding the locations of fishing vessels were acquired from the Marin AIS data service website (https://www.marinetraffic.com, accessed on 1 March 2020 to AIS data service (https://www.marinetraffic.com, accessed 1 March 20 28 February 2023) forwebsite the period spanning from March 2020 to February 2023. Theon vessels’ February 2023) for the period spanning from March 2020a screenshot to February 2023. The positions were obtained through screenshots taken at 9:00 UTC, with interval of one day. The AIS data service website marks the vessel positions with arrow-shaped positions were obtained through screenshots taken at 9:00 UTC, with a screenshot symbols. In this study, threshold method was used to extract these symbols, and the arrow of one day. The AISa data service website marks the vessel positions with pixel coordinates of the centers of these symbols in the captured images were obtained. symbols. this study, threshold method the was used to extract symbols, These pixel In coordinates wereathen used to determine vessel positions in thethese captured pixel coordinates of the centers of these symbols in the captured images images. Leveraging the tile pyramid model [26], the pixel coordinates were converted to were o latitude and longitude coordinates obtainused the geographic positions each fishing vessel.in the c These pixel coordinates weretothen to determine theofvessel positions These coordinates were then imported into Matlab and ArcGIS software (Matlab R2021a images. Leveraging the tile pyramid model [26], the pixel coordinates were conv and ArcMap 10.4.1) to analyze the spatial–temporal distribution of fishing vessels. latitude and longitude coordinates to obtain the geographic positions of each fish Sea Surface Temperature (SST) is a critical oceanographic component influencing sel. These ofcoordinates were then imported Matlab and ArcGIS and software the creation fishing grounds since it directly affects into the migration, reproduction, R2021a and ArcMap 10.4.1) [27]. to analyze the spatial–temporal distribution spawning of marine organisms Chlorophyll a concentration (Chl-a) also playsofa fishing significant role in influencing the distribution upper-layer fishery resources [28]. Thisinfluen Sea Surface Temperature (SST) is aofcritical oceanographic component study investigated the link between the marine environment and the spatial–temporal creation of fishing grounds since it directly affects the migration, reproducti distribution of fishing vessels using data on SST and Chl-a concentration. SST and Chl-a spawning ofdata marine organisms [27].and Chlorophyll concentration (Chl-a) concentration were provided by SST Chlor_a L3Badata products from NASA’salso pla nificant website role in(https://www.earthdata.nasa.gov, influencing the distributionaccessed of upper-layer fishery [2 Earthdata on June 2020 to May resources 2023), which obtained by the Resolution Spectrometer (MODIS) board studywere investigated theModerate link between theImaging marine environment andonthe spatial–te the Aqua satellite, with a temporal resolution of one month and a spatial resolution of SST an distribution of fishing vessels using data on SST and Chl-a concentration. concentration data were provided by SST and Chlor_a L3B data products from Earthdata website (https://www.earthdata.nasa.gov, accessed on June 2020 to Ma which were obtained by the Moderate Resolution Imaging Spectrometer (MO board the Aqua satellite, with a temporal resolution of one month and a spatial re Sensors 2024, 24, 781 4 of 19 4 km. The data used in this study span the period from March 2020 to February 2023. The SST L3B data product is based on the method of the split-window nonlinear SST (NLSST) algorithm [29], and the Chlor_a L3B data product is based on the algorithm of Hu et al. [30], which combines the empirical band-difference method at low Chl-a concentrations and the ratio-transformation method at higher Chl-a concentrations. 2.2. Methods 2.2.1. Characterization Indicators and Calculation Methods of the Spatial–Temporal Distribution Characteristics of Fishing Vessels Based on the acquired AIS data, the interannual distribution heat of fishing vessels and the monthly and seasonal variations in fishing vessel numbers were selected as research indicators to analyze the spatial–temporal distribution of fishing vessels in the study area. 1. Normalized Heat Index In order to depict the interannual spatial distribution of fishing vessels in the northern Indian Ocean from March 2020 to February 2023, this study presents interannual spatial distribution heat maps of fishing vessels in the study area. The map production process is as follows: First, the study area was divided into 10,064 (136 × 74) equally sized 0.5◦ × 0.5◦ grids. Using these 0.5◦ × 0.5◦ grids as the statistical unit, the annual numbers of fishing vessels in each grid were calculated. Then, the Normalized Heat Index (NHI) was obtained by normalizing the vessel numbers in each grid [31]. The NHI is used as the indicator to create the interannual spatial distribution heat maps of fishing vessels in the study area. The calculation formula of NHI is as follows: NH Ii,j = Ni,j − Min Max − Min (1) where NHIi,j is the NHI for grid j in year i, Ni,j is the number of fishing vessels in grid j in year i, and Max and Min are the maximum and minimum values of fishing vessels numbers across all 0.5◦ × 0.5◦ grids for the three years, respectively. The range of NHIi,j is from 0 to 1, with a higher NHIi,j approaching 1 indicating a ‘hot’ grid in year i, reflecting more frequent fishing operations in the corresponding waters. Conversely, a lower NHIi,j approaching 0 indicates a ‘cold’ grid, indicating fewer fishing operations. 2. Measurement method of time variations of fishing vessel numbers The number of fishing vessels in a specific time period can also reflect the intensity of fishing operations. The greater number of fishing vessels presents stronger fishing operations. The statistical model for the number of fishing vessels is n F= ∑ Fi (2) i =1 where F is the number of fishing vessels in a specific time period, Fi is the number of fishing vessels at moment i, and n represents time. For the convenience of analysis, this study calculated the number of fishing vessels with months and seasons as time units to measure the intensity of fishing operations in the northern Indian Ocean. 2.2.2. Monthly Spatial Distribution Statistics of Fishing Vessels In order to clarify the seasonal variation characteristics of fishing vessels’ spatial distribution in the study area, monthly AIS data were counted at a spatial resolution of 1◦ × 1◦ , and the monthly numbers of fishing vessels in each grid were obtained. To mitigate the impact of abnormal values and other factors, the medians of fishing vessel numbers in the same grid location and month from 2020 to 2023 were used as Catch Per Unit Effort (CPUE) in this grid [32]. Due to the presence of small numbers of fishing vessels in some grids, this study calculated the quartiles (Q1~Q3) of CPUE [33]. Using Q3 (the third quartile) as the threshold, grids with CPUEs higher than Q3 were defined as fishing Sensors 2024, 24, 781 5 of 19 operation areas in the northern Indian Ocean. These grids were visually represented and analyzed. From March 2020 to February 2023, there were 8456 grids with CPUEs higher than Q3, which were counted monthly in the 1◦ × 1◦ grids, with quartile values as follows: Q1 = 2, Q2 = 6, Q3 = 15. 2.2.3. Oceanographic Factor Spatial Analysis Monthly SST and Ch-a concentration data from March 2020 to February 2023 were also used in this study. The monthly median values of SST and Chl-a concentration were computed for the same month and the same latitude and longitude positions over the three years, and the monthly spatial distributions of SST and Chl-a concentration of the study area were obtained. Subsequently, these distributions were overlaid with fishing operation areas, and the monthly spatial distribution maps for fishing operation areas with SST and Chl-a concentration were created, which could facilitate the qualitative analysis of the relationship between fishing operations and oceanographic factors. Finally, the quantitative analysis was conducted by extracting the corresponding oceanographic factor values based on the latitude and longitude coordinates of the fishing vessels. The variation ranges of oceanographic factors in fishing operation areas were identified and the Analysis of Variance (ANOVA) was used to compare the extracted data. 2.2.4. Oceanographic Factor Preferred Range Calculation Frequency analysis and the empirical cumulative distribution function (ECDF) [34–36] were used to calculate the preferred ranges of two oceanographic factors (SST and Chl-a concentration) for fishing operations in the northern Indian Ocean. Firstly, the means, standard deviations, and preferred ranges (means ± standard deviations) of SST and Chl-a concentration for fishing operation areas in the study area were computed. Subsequently, the association between the oceanographic factors and CPUEs during the same period was analyzed by ECDF, and the CPUE-weighted preferred ranges of SST and Chl-a concentration were computed (factor values at the maximum D(t) ± standard deviations). The intersection of the two ranges represents the optimum SST and Chl-a concentration for fishing operations in the study area. In this analysis, three functions were used as follows:  n 1, xi ≤ t 1 F ( t ) = n ∑ l ( x i ), l ( x i ) = 0, xi > t i =1 n y (3) G (t) = n1 ∑ yi l ( xi ) i =1 D (t) = | F (t) − G (t)| where F(t) is the empirical cumulative distribution function, G(t) is the CPUE-weighted cumulative distribution function, l(xi ) is the indication function, and D(t) is the absolute value of the difference between the two curves F(t) and G(t) at any point t. n is the number of fishing vessels, xi is the oceanographic factor of the fishing vessel i, t is the index ranking the ordered observations from the lowest to highest values of the oceanographic factors, yi is the CPUE of the fishing vessel i, and y is the mean of CPUE for all fishing vessels. In the above work, the calculation and statistics were completed using Matlab R2021a software, and the heat maps and monthly spatial distribution maps were produced with ArcMap 10.4.1 software. 3. Results 3.1. Statistics of Fishing Vessels Figure 2 shows the interannual distribution heat maps of fishing vessels in the northern Indian Ocean. It is evident that fishing vessels are highly concentrated in the surrounding areas of the EEZ of the Maldives, the Palk Strait, the coastal areas of India, the Seychelles, and the Gulf of Bahrain. Fishing vessels are mostly active in fishing grounds and aquaculture areas, presenting an overall clustered distribution. Over the three years, the heat of Sensors 2024, 24, 781 Sensors 2024, 24, x FOR PEER REVIEW 6 of 19 6 of 19 fishing vessels increased annually. In addition to the mentioned regions, the heat of fishing vessels increased in the coastal areasto ofthe Iran, Pakistan,regions, Myanmar, and fishing gradually vessels increased annually. In addition mentioned the Thailand, heat of fishing the central Arabian Sea. There are also areas with consistently low fishing vessel heat vessels gradually increased in the coastal areas of Iran, Pakistan, Myanmar, Thailand, and in studyArabian area, such the Red Sea,areas the Gulf Aden, thelow northern southeastern thethe central Sea.as There are also withof consistently fishingand vessel heat in the areas of the Bay of Bengal, the Andaman Sea, the eastern Indian Ocean, the EEZareas of the study area, such as the Red Sea, the Gulf of Aden, the northern and southeastern of Maldives, and the British Indian Ocean Territory. Furthermore, in the center of the Bay the Bay of Bengal, the Andaman Sea, the eastern Indian Ocean, the EEZ of the Maldives, of Bengal, fishing vessels exhibit a strip-like distribution, with heat first and and the British Indian Ocean Territory. Furthermore, in the center of theincreasing Bay of Bengal, fishthen decreasing. ing vessels exhibit a strip-like distribution, with heat increasing first and then decreasing. Figure2.2.Interannual Interannualdistribution distributionheat heatmaps mapsof offishing fishingvessels. vessels. Figure Figure Figure 3a 3a shows shows the the monthly monthly variation variation lines lines chart chart of of the the total total fishing fishing vessels vessels in in the the study to 2023. 2023.InInthree threeyears, years, the number fishing vessels drastically study area area from 2020 to the number of of fishing vessels drastically dedeclines after March around September. Throughout the from period from clines after March andand thenthen risesrises around September. Throughout the period October October to March of the next year, the numbers of fishing vessels remain relatively to March of the next year, the numbers of fishing vessels remain relatively high,high, with with March October exhibiting counts higher adjacent months (February March and and October exhibiting counts higher thanthan theirtheir adjacent months (February and and April, September November, respectively). April, September andand November, respectively). The monthly numbers of fishing vessels exhibit periodic variations from March 2020 to February 2023, and the trend analysis of the total numbers of fishing vessels increases Sensors 2024, 24, x FOR PEER REVIEW 7 of 19 markedly (Figure 3b, where the trend line is represented in red, and the relevant parame7 of 19 ters are shown in Table 1, p < 0.05). Sensors 2024, 24, 781 Figure3.3.Monthly Monthly variations trend analysis the numbers of fishing (a) Monthly Figure variations andand trend analysis of theofnumbers of fishing vessels.vessels. (a) Monthly variavariations. (b) Trend analysis. The averages and trend lines are shown in red. tions. (b) Trend analysis. The averages and trend lines are shown in red. The monthly numbers of fishing vessels exhibit periodic variations from March 2020 to February 2023, and the trend analysis of the total numbers of fishing vessels increases Slope 3b, where the trend line Intercept R2 markedly (Figure is represented in red, and the relevant parameters are shown 268.09 in Table 1, p < 0.05). 6941.11 0.60 Table 1. Linear regression of monthly variations of the numbers of fishing vessels. Table 1. Linear regression of monthly variations of the numbers fishing vessels. According to the averages of fishing vessel numbers inofdifferent seasons and the rela- Time 03.2020–02.2021 Time 03.2021–02.2022 03.2022–02.2023 03.2020–02.2023 03.2020–02.2021 03.2021–02.2022 03.2022–02.2023 03.2020–02.2023 tive variation Slope rates of the annual averages in the study area, the fishing vesselRnumbers have 2 Intercept obvious seasonal differences, showing the characteristics of winter > autumn > spring > sum268.09 6941.11 0.60 mer (Table 2). Over the three years, the number of fishing vessels from March 2020 to February 2021 is lower than the following two years, with a negative relative variation rate in According to(fishing the averages of fishing vessel numbers in different seasons and the relative the spring of 2021 season). variation rates of the annual averages in the study area, the fishing vessel numbers have obvious2.seasonal showing characteristics of winter > autumn > spring > summer Table Seasonaldifferences, variation statistics of the the numbers of fishing vessels. (Table 2). Over the three years, the number of fishing vessels from March 2020 to February Summer Winter 2021 isSpring lower than the following two years, with aAutumn negative relative variation rate in the Year (March to (June to (September to (December to spring of 2021 (fishing season). May) August) November) February) Table 2. Seasonal variation statistics of the numbers of fishingRelative vessels. Relative Relative Relative Average/ Average/ Average/ Average/ Average/ Variation Variation Variation Variation Spring Summer Month Month Month Month Month Winter Autumn Rate/% Rate/% Rate/% Year (March to (June toRate/% (December to February) (September to November) August) -18.24 8681 9794May) 12.82 7098 8799 1.36 9034 4.07 Relative Relative Relative Relative 11,859 10,049 -15.26 9117 -23.12 12,334 4.01 15,935 34.37 Average/ Average/ Average/ Average/ Average/ Variation Variation Variation Variation Month Month Month Month 15,163 Month 15,865 4.63 11,914 -21.43 17,326 14.26 15,545 2.52 Rate/% Rate/% Rate/% Rate/% 11,901 11,903 0.02 9376 -21.22 12,820 7.72 13,505 13.48 8681 11,859 15,163 11,901 9794 12.82 10,049 −15.26 15,865 4.63 3.2. Monthly Spatial 11,903 0.02 7098 9117 11,914 Distribution 9376 −18.24 8799 1.36 −23.12 12,334 4.01 −21.43 17,326 14.26 Characteristics of Fishing Vessels −21.22 12,820 7.72 9034 15,935 15,545 13,505 4.07 34.37 2.52 13.48 Figure 4 shows the monthly distribution of fishing operation areas in the northern Indian Ocean. It is evident that the latitudinal positions of fishing operation areas (CPUE > 3.2. Monthly Spatial Distribution Characteristics of Fishing Vessels Q3) mainly concentrate between 20° N and 6° S, and the longitudinal positions are mainly Figure shows monthly distribution of Africa. fishingThere operation areas in the northlocated in the4 west of the 90° E to the eastern coast of are also some scattered ern Indian Ocean. It is evident that the During latitudinal of fishingperiod operation areas fishing operation areas outside this range. the positions northeast monsoon (October ◦ N and 6◦ S, and the longitudinal positions (CPUE > Q3) mainly concentrate between 20 to March), there are more fishing operation areas located in the mentioned range, while are mainly located in monsoon the west of 90◦ E (April to the eastern coast of fishing Africa. operation There are also during the southwest period to September), areassome are scattered fishing operation areas outside this range. During the northeast monsoon period fewer and scattered. (October to March), there are more fishing operation areas located in the mentioned range, while during the southwest monsoon period (April to September), fishing operation areas are fewer and scattered. CPUEs in the Gulf of Aden, the Persian Gulf, the Gulf of Oman, and the northern Arabian Sea are generally low and there is no CPUE over Q3 in the Red Sea all through the Sensors 2024, 24, x FOR PEER REVIEW Sensors 2024, 24, 781 8 of 19 8 of 19 CPUEs in the Gulf of Aden, the Persian Gulf, the Gulf of Oman, and the northern Arabian Sea are generally low and there is no CPUE over Q3 in the Red Sea all through the year. High High CPUEs CPUEs are observed in the Bay of Bengal during both the northeast year. northeast monsoon monsoon (December to March) and (December and southwest southwestmonsoon monsoon(June (JunetotoSeptember) September)periods. periods. From October distribution range, range, extending extending October to March, fishing vessels exhibit a broad distribution from The CPUE CPUE remains remains from the the central central Arabian Sea to the west of the EEZ of the Maldives. Maldives. The relatively during the the period period from from October October to to December, December, showing showing an an increase increase relatively consistent consistent during compared distribution range range of of fishing fishing vessels vessels compared to the months from January to March. The distribution narrows from April April to toMay, May, leading leadingtotoa adecline declineininCPUE. CPUE. CPUE reaches minimum narrows from CPUE reaches its its minimum in in June to July gradually from August to September. area clear exhibits clear June to July andand gradually rises rises from August to September. This areaThis exhibits seasonal seasonal in the distribution of fishing making the most significant variationsvariations in the distribution of fishing vessels,vessels, making it the itmost significant area area for for variations in fishing vessels distribution within the study variations in fishing vessels distribution within the study area. area. Fishing operation operationareas areasare areobserved observed EEZ of Somalia from February to June inin thethe EEZ of Somalia from February to June and and from September to November. These areas are mainly concentrated in the south of from September to November. These areas are mainly concentrated in the south of Ras Ras Hafun from February to June, with increasing CPUEs from February to March and Hafun from February to June, with increasing CPUEs from February to March and decreasing Fishing operation operation areas areas are are mostly mostly focused focused in in the the waters waters decreasing from April to June. Fishing between Alula and and Bayla Bayla(Somalia) (Somalia)from fromSeptember September November, with a higher CPUE between Alula toto November, with a higher CPUE in in October. October. In the waters of of Sri Sri Lanka Lanka to to 6° 6◦ SS in the Indian Ocean, CPUE CPUE remains remains the southwestern southwestern waters Indian Ocean, consistently high allthrough the year. From June to December, an increase in fishing consistently high allthrough the year. From June to December, an increase in fishing operation areas is noted in the eastern Indian Ocean, but CPUE is relatively low. From operation areas is noted in the eastern Indian Ocean, but CPUE is relatively low. From January to May, there is generally no fishing operation in this waters. January to May, there is generally no fishing operation in this waters. Figure 4. Cont. Sensors2024, 2024,24, 24,781 x FOR PEER REVIEW Sensors 9 9ofof19 19 Figure 4. Monthly spatial distribution of fishing vessels and SST. Figure 4. Monthly spatial distribution of fishing vessels and SST. 3.3. 3.3. Fishing Fishing Operations Operations with SST The It is is observed observed The monthly monthly spatial spatial distributions of SST are also shown in Figure 4. It ◦ N~6 ◦ S), there that thewaters waterswhere wherefishing fishing vessels concentrated is awarm large that in the vessels areare concentrated (20°(20 N~6° S), there is a large warm area extending from the eastern of the Indian to the eastern of water water area extending from the eastern of the Indian Ocean Ocean to the eastern coast ofcoast Africa Africa from January to March, leading to increase an increase in CPUE. During Apriland andMay, May,SST SST from January to March, leading to an in CPUE. During April Sensors 2024, 24, 781 10 of 19 reaches its peak (mostly over 29 ◦ C), leading to a subsequent decrease in CPUE. From June to August, the high SST area (over 29 ◦ C) contracts, contributing to a further decrease in CPUE. SST rises in October relative to September as the southwest monsoon down, which leads to a rise in CPUE. By November, SST becomes relatively uniform, contributing to a decrease in CPUE. In December, the high-SST area shifts southward, leading to an increase in CPUE in the western area of the EEZ of the Maldives. From June to September, the eastern coast of Africa is influenced by cold currents, resulting in a lower SST (24 to 27 ◦ C) compared to other months. In this period, the low SST area shows a distinct band-shaped area and without fishing operation area. The SST ranges of the fishing operations areas in each season were calculated, also with the means and variances. The ANOVA was used to test the seasonal effects. All results are shown in Table 3. Table 3. Statistics and variance analysis of SST of fishing operation areas in each season. Year Season Min (◦ C) Max (◦ C) Mean (◦ C) Var MSB MSW F 03.2020–02.2021 Spring Summer Autumn Winter 20.26 24.56 25.56 18.41 34.56 35.80 34.41 32.93 29.96 29.75 29.33 28.64 1.74 1.59 0.74 1.49 5268.02 1.42 3708.29 03.2021–02.2022 Spring Summer Autumn Winter 20.68 25.04 23.62 18.10 33.76 35.75 33.24 32.50 29.81 29.22 29.10 28.63 1.44 0.93 0.69 1.50 5549.84 1.17 4741.54 03.2022–02.2023 Spring Summer Autumn Winter 20.37 24.68 24.17 18.62 33.62 35.77 33.54 31.23 29.72 29.14 28.81 28.38 1.57 1.13 0.54 1.25 8684.58 1.07 8105.29 It can be seen that over the three years, the average SST ranges of the fishing operation areas in the four seasons are as follows: 20.44 to 33.98 ◦ C in spring, 24.76 to 35.77 ◦ C in summer, 24.45 to 33.73 ◦ C in autumn, and 18.38 to 32.22 ◦ C in winter. The means show the characteristic order of spring > summer > autumn > winter. In the variance analysis of each year, the Mean Squares Between (MSB) are much higher than the Mean Squares Within (MSW). At the significance level of α = 0.05, the F-values are also much higher than the critical value (Fcrit = 2.60), indicating a significant seasonal impact on the SST of the fishing operation areas. 3.4. Fishing Operations with Chl-a Concentration Monthly spatial distributions of fishing vessels and Chl-a concentration are shown in Figure 5. It is observed that, under the influence of the monsoon, Chl-a concentration in the Arabian Sea and the eastern coast of Africa varies significantly, whereas other areas exhibit relatively stable conditions. In the Arabian Sea, the high Chl-a concentration area generally exhibits a latitudinal pattern during the northeast monsoon period. It extends southward from the northern Arabian Sea in December and contracts northward in March. Fishing vessels exhibit movements northward and southward in response to changes in the latitudinal position of areas with high Chl-a concentration and are mainly distributed between the high- and low-concentration areas. In the eastern coast of Africa, Chl-a concentration gradually increases from south to north during the southwest monsoon period. The high concentration area could extend to the central Arabian Sea. Conversely, during the northeast monsoon period, Chl-a concentration increases from north to south, with the high-concentration area extending to the coast of Kenya. From February to June, fishing vessels in the south of Ras Hafun are concentrated in both mid-value and low-value areas of Chl-a concentration. From Sensors 2024, 24, x FOR PEER REVIEW Sensors 2024, 24, 781 11 of 19 11 of 19 to the coast of Kenya. From February to June, fishing vessels in the south of Ras Hafun are concentrated in both mid-value and low-value areas of Chl-a concentration. From September fishing vessels in thein waters Alula to Bayla September totoNovember, November, fishing vessels the from waters from Alulaaretoconcentrated Bayla are in the mid-value area of Chl-a concentration. concentrated in the mid-value area of Chl-a concentration. Figure 5. Cont. Sensors2024, 2024,24, 24,781 x FOR PEER REVIEW Sensors 1212ofof19 19 Figure 5. 5. Monthly Monthly spatial spatial distribution distribution of of fishing fishing vessels vessels and Figure and Chl-a Chl-a concentration. concentration. Table 44 shows shows the the Chl-a Chl-a concentration concentration ranges means, variances, Table variances, and and variance variance analysis analysis results of of fishing fishing operations operations areas in each season. Over results Over the the three years, years, the the average average Chl-a Chl-a concentration ranges concentration ranges of the fishing operation operation areas areas for for each each season season are are as as follows: follows: 0.05 0.05 to to 3 in autumn, 3 in3 summer, 3 in autumn, 19.79 mg/m mg/m33ininspring, 0.040.04 to 87.89 mg/m and 19.79 spring,0.05 0.05toto69.10 69.10mg/m mg/m in summer, to 87.89 mg/m 3 in winter. 3 in winter. 0.05 0.05 to 23.70 mg/mmg/m The means in summer and autumn are higher those in and to 23.70 The means in summer and autumn arethan higher than springinand winter. those spring and winter. Table 4. Statistics and variance analysis of Chl-a concentration of fishing areas in each seaTable Statistics and variance analysis of Chl-a concentration of operation fishing operation areas in son. each season. Year Year 03.2020–02.2021 03.2020–02.2021 03.2021–02.2022 03.2021–02.2022 03.2022–02.2023 03.2022–02.2023 Season Min (mg/m3) Min Season Spring 0.04 (mg/m3 ) Summer 0.050.04 Spring Autumn 0.050.05 Summer Autumn Winter 0.060.05 Winter Spring 0.050.06 Spring Summer 0.060.05 Summer Autumn 0.050.06 Autumn 0.05 Winter 0.060.06 Winter Spring 0.05 Spring 0.05 Summer 0.040.04 Summer Autumn Autumn 0.030.03 Winter Winter 0.030.03 Max (mg/m3) Mean (mg/m3) Var MSB MSW Max Mean Var MSB MSW 3) 20.303 ) 0.62 (mg/m (mg/m0.26 73.04 0.86 13.98 20.30 0.26 0.62 1263.56 4.33 96.54 73.04 0.86 0.79 13.98 6.27 1263.56 4.33 96.54 0.79 0.29 6.27 0.27 19.56 19.56 0.29 0.20 0.27 0.17 12.19 12.19 0.20 0.17 2.53 50.36 0.48 1102.74 2.64 50.36 0.48 2.53 86.98 0.77 7.14 1102.74 2.64 86.98 0.77 7.14 28.75 28.75 0.39 0.39 0.99 0.99 26.89 0.38 1.11 26.89 0.38 1.11 83.91 83.91 0.53 0.53 5.16 5.16 285.41 2.60 2.60 285.41 80.16 0.50 3.16 3.16 80.16 0.50 22.79 0.29 0.48 22.79 0.29 0.48 F F 291.80 291.80 417.70 417.70 109.91 109.91 In the variance analysis of each year, the MSB is also much higher than the MSW. At the significance level of α = 0.05, the F-values are also much higher than the critical value (Fcrit = 2.60). Compared with SST, the Chl-a concentration of the fishing operation areas Sensors 2024, 24, x FOR PEER REVIEW s 2024, 24, x FOR PEER REVIEW Sensors 2024, 24, 781 13 of 19 13 of 19 In the variance analysis of each year, the MSB is also much higher than the MSW. At 13 of 19 In variance analysis the MSB are is also Atvalue thethe significance level of of α =each 0.05,year, the F-values alsomuch muchhigher higherthan thanthe theMSW. critical the significance level of α = 0.05, the F-values are also much higher than the critical value (Fcrit = 2.60). Compared with SST, the Chl-a concentration of the fishing operation areas (Fcrit =with 2.60). Compared with SST, MSW the Chl-a concentration of the than fishing operation areas that lower and higher lowerinF-values SST. This indicates withMSB lower MSB and higherresult MSWinresult lower F-values than SST. This indicates that with the lower MSB and higher MSW in lower F-values than This indicates that by Chl-a concentration of the result fishing areas in the SST. study is less the Chl-a concentration of the operation fishing operation areas in thearea study areaaffected is less affected by the Chl-a concentration of the fishing operation areas in the study area is less affected by seasonal variations compared with SST. seasonal variations compared with SST. seasonal variations compared with SST. 3.5. Oceanographic Factor Preferred 3.5. Oceanographic Factor Preferred Ranges Ranges 3.5. Oceanographic Factor Preferred Ranges The ranges of SST and Chl-a concentration for fishing the northern The ranges of SST and Chl-a concentration for fishing operations inoperations the northerninIndian ◦ 3 The ranges of SST and Chl-a concentration for fishing operations in the northern Indian 3, respectively Indian are°C18.09 35.80 C and 0.03 to 96.54 mg/m , respectively (Figure 6). The Ocean are 18.09Ocean to 35.80 and to 0.03 to 96.54 mg/m (Figure 6). The frequency 3, respectively (Figure 6). The frequency Oceandistribution are 18.09 to 35.80 °C and of 0.03SST/Chl-a to 96.54 mg/m frequency distribution histograms of SST/Chl-a concentration CPUE a skewed histograms concentration with CPUE with exhibit a exhibit skewed ◦ C and distribution histograms of SST/Chl-a concentration with CPUE exhibit a skewed distribution trend. The mean of SST is 29.15 the standard deviation is 1.19, distribution trend. The mean of SST is 29.15 °C and the standard deviation is 1.19, while the while 3 with distribution The mean of SST is 29.15 °C3and the standard deviation isof1.19, the of 1.73. By the mean of Chl-a concentration is 0.46 the standard deviation mean oftrend. Chl-a concentration is 0.46 mg/m with themg/m standard deviation 1.73.while By frequency 3 meananalysis, of Chl-afrequency concentration is 0.46 mg/m with the standard deviation of 1.73. By frequency analysis, approximately 74.70% of fishing vessels are concentrated in the area approximately 74.70% of fishing vessels are concentrated in the area where SST ◦ C (29.15 ◦in analysis, approximately 74.70% of fishing vessels are concentrated the area where SST where SST ranges from 27.96 to 30.34 ± 1.19 C), and 96.21% are concentrated in ranges from 27.96 to 30.34 °C (29.15 ± 1.19 °C), and 96.21% are concentrated in the area where 3 (0.46 3 ). ranges from 27.96 to 30.34 °C (29.15 ± 1.19 °C), and 96.21% are concentrated in the area where the area where Chl-a concentration ranges from 0.03 to 2.19 mg/m ± 1.73 mg/m Chl-a concentration ranges from 0.03 to 2.19 mg/m3 (0.46 ± 1.73 mg/m3). Chl-a concentration ranges from 0.03 to 2.19 mg/m3 (0.46 ± 1.73 mg/m3). Figure 6. Frequency distribution histograms of SST/Chl-a concentration with CPUE. (a) SST. (b) Chl-a Figure 6. Frequency distribution histograms of SST/Chl-a concentration Figureconcentration. 6. Frequency distribution histograms of SST/Chl-a concentration with CPUE. (a) SST. (b)with Chl-aCPUE. (a) SST. concentration. (b) Chl-a concentration. The ECDF results are shown inshown Figurein7.Figure The maximum D(t) values correTheanalysis ECDF analysis results 7. The maximum D(t) values corThe ECDF to analysis results are shown in are Figure 7. The maximum D(t) values corre3, respectively, sponding SST and Chl-a concentration occur at 28.28 °C and 0.08 mg/m ◦ 3 , respecresponding to SST and Chl-a concentration occur at 28.28 C and 0.08 mg/m 3 sponding to SST and their Chl-arespective concentration occur at 28.28 °C and 0.08mg/m mg/m respectively, 3). , Then, different from means (29.15 °C and 0.46 the CPUE◦ 3 tively, frommeans their respective means (29.15 C and 0.46 mg/m ). Then, the CPUE3). Then, different from theirdifferent respective (29.15 °Cconcentration and 0.46 mg/m the weighted preferred ranges of SST and Chl-a are obtained: 27.09CPUEto 29.47to °C29.47 ◦ C weighted preferred ranges of SST and Chl-a concentration are obtained: weighted preferred ranges of SST andmg/m Chl-a3 concentration are 3obtained: 27.09 to 29.4727.09 °C ◦ 3 3 (28.28 ± (28.28 1.19 °C) and 0.03 to 1.81 (0.08 ± 1.73 mg/m ), respectively. 1.19toC) and 0.033 to 1.81± mg/m (0.08 1.73 mg/m ), respectively. 3), ± (28.28 ± 1.19 °C) and±0.03 1.81 mg/m (0.08 1.73 mg/m respectively. Figure 7. Empirical cumulative distribution function for CPUE and SST (a), Chl-a concentration Figure(b). 7. Empirical cumulative distribution function for CPUE and SST (a), Chl-a concentration Figure 7. Empirical cumulative distribution function for CPUE and SST (a), Chl-a concentration (b). (b). Combining the results of frequency analysis and ECDF, intersecting two preferred ranges, the optimum ranges of SST and Chl-a concentration for fishing operations in the northern Indian Ocean are determined as 27.96 to 29.47 ◦ C and 0.03 to 1.81 mg/m3 . Sensors 2024, 24, 781 14 of 19 4. Discussion 4.1. The Numbers and Spatial–Temporal Distribution Variations of Fishing Vessels The winter northeast monsoon and summer southwest monsoon have significant impacts on the distribution of fishing vessels in the northern Indian Ocean [37]. The northeast monsoon periodis characterized by weaker wind (mostly at grade 3 to 4), dry and clear weather, and good visibility on the sea surface, making this period suitable for fishing operations. On the other hand, during the summer monsoon period, the wind gets stronger (reaching up to grade 6 to 7 in the Arabian Sea and up to grade 10 in its western area), also with humid and rainy weather and decreased visibility, making this period unsuitable for fishing operations [38]. Additionally, the spawning period for many catch targets occurs in summer. The summer southwest monsoon induces upwellings, transferring nutrients from the lower layers to the surface waters and creating favorable conditions for the reproduction of the catch targets. The reduced fishing operations during this period are beneficial for minimizing disturbances to the breeding activities and promoting the growth and development of the catch targets [39]. Therefore, the northeast monsoon period (October to March) is often the fishing season, with widespread distribution and frequent fishing operations, while the southwest monsoon period (April to September) is considered the fishing moratorium, with few fishing vessels distributed. In the southwest monsoon period, a majority of fishing vessels typically return. The distribution of fishing vessels decreases in the Arabian Sea along with its adjacent high sea of the Indian Ocean, while a few vessels navigate to areas less affected by the southwest monsoon in the eastern Indian Ocean for fishing operations, such as the Bay of Bengal and its adjacent waters [40]. This shift could contribute to high CPUEs in this area compared with the period from November to March (fishing season). As the southwest monsoon gradually subsides in September, fishing vessels start returning to the operation areas. Consequently, the distribution of fishing vessels in this month appears in a strip-like pattern, as seen in the Bay of Bengal and the eastern Indian Ocean. In October, as the fishing season commences and some fishing vessels are still in the process of relocating, the CPUE of the south waters of India reaches its peak for the entire year. The high fishing vessel numbers in March and October compared with their adjacent months (February and April, September and November, respectively) are likely related to the end and beginning of the fishing season. Furthermore, a rise in fishing vessels in March could be related to the warmer weather [41]. Due to the impact of the COVID19 pandemic [42–45], the number of fishing vessels was relatively low in 2020 (Table 2). As countries gradually lifted pandemic control measures in 2021 [46], fishing operations resumed and the number of fishing vessels began to recover, which also led to a significant increase during the fishing season (December 2021 to February 2022). The monthly numbers of fishing vessels showed an upward trend from 2020 to 2023 (Figure 3). The underlying reasons for this trend, aside from the impact of the COVID-19 pandemic, also include national fishery management policies [5], the abundance of fishery resources [47], and the upgrading of the data acquisition website. Nevertheless, specific reasons behind this trend necessitate further research for a comprehensive understanding. 4.2. The Impacts of Oceanographic Factors on Fishing Operations According to the CPUE spatial–temporal distribution maps (Figures 4 and 5), there is a significant difference in CPUE between the northeast monsoon and the southwest monsoon period. CPUE increases during the northeast monsoon, while it decreases during the southwest monsoon. CPUE is closely related to the spawning and migration of fishery resources [48] and is also influenced by the seasonal variations due to monsoons. Seasonal variations in SST and Chl-a concentration caused by the monsoons eventually affect the distribution of fishing grounds. SST is a key oceanographic factor affecting the distribution of fishing grounds, which could indicate the distribution and variation of fishing grounds. From April to May, in the fishing operation areas between 20◦ N and 6◦ S, SST is mostly above 29 ◦ C due Sensors 2024, 24, 781 15 of 19 to continuous sunlight exposure. From June to September, the combined effects of the southwest monsoon, typhoons, surface water mixing, evaporation, and surface wind stress reduce the heat of sea surface water, leading to a decrease in SST [49]. This period also witnesses the influence of the Somali Current, resulting in the appearance of a low-SST area (24 to 27 ◦ C) along the east coast of Africa and the western Arabian Sea. CPUE is relatively low in these months, suggesting that, aside from the impacts of the fishing moratorium and monsoon, excessively high or low SSTs can also contribute to a decrease in CPUE. From October to March, SST in the area between 20◦ N and 6◦ S is mostly concentrated in the range of 26 to 30 ◦ C. Therefore, during the southwest monsoon period, the SST in fishing operation areas varies significantly, whereas during the northeast monsoon period, the SST remains relatively stable. The analysis indicates that the suitable SST during the northeast monsoon period is identified as an environmental factor contributing to high CPUE in the fishing operation areas. The optimum SST range for fishing operations in the northern Indian Ocean obtained in this study is 27.96 to 29.47 ◦ C. This range is generally lower than the optimum SST range of 29.3 to 30.8 ◦ C reported by Xu et al. [50]. The difference in results could be attributed to the different study years. Xu et al.’s study took place during an El Niño year (2016), which led to an increase in SST [51]. The dynamic nature of oceanographic conditions underscores the importance of considering temporal variations in assessing the optimum SST for fishing operations. Chl-a concentration, representing the content of bait and plankton, is also used for predicting fishing ground locations [52]. Seasonal variations in Chl-a concentration are seen in the Arabian Sea and the eastern coast of Africa. Chl-a concentration is generally high all through the year in the northern Arabian Sea, especially in the Gulf of Oman and its adjacent waters [53]. In the southern Arabian Sea, Chl-a concentration decreases, while in the central waters, it remains in an intermediate range between high and low values. This distribution pattern could be a contributing factor to the distribution of fishing operation areas in the central Arabian Sea. In the eastern coast of Africa, fishing operation areas are concentrated in the areas with both mid-range and low-range Chl-a concentrations during the northeast monsoon period. However, from June to September, there is a low distribution of fishing vessels in this area. Aside from the impacts of the southwest monsoon and fishing moratorium, the low-temperature and high-Chl-a concentration area formed by cold currents and upwellings may inhibit fishery resources, leading to a decrease in CPUE. Additionally, there are fishing operation areas in coastal waters with high Chl-a concentration, such as the coastal areas of Pakistan and Cape Comorin (India). In summary, the impact of Chl-a concentration on fishing operations in the study area lacks a standardized criterion, which indicates that the relationship between fishing operations and Chl-a concentration is not straightforward. In order to understand the relationship comprehensively, more detailed fishery resource data, such as the biological characteristics of the catch targets, are necessary for future research. The optimum Chl-a concentration range for fishing operations in the northern Indian Ocean obtained in this study is 0.03 to 1.81 mg/m3 , which differs from the optimum range of 0.2 to 0.5 mg/m3 reported by Yanget al. [54]. The difference in results could be attributed to differences in the study areas, considering coastal areas or not. 4.3. Speculations on the Locations and Seasonal Variations of Fishing Grounds in the Northern Indian Ocean Based on the spatial–temporal distribution of fishing vessels in the study area, in addition to coastal waters and EEZs, there are also fishing grounds in high seas. Preliminary inferences suggest the presence of fishing grounds in the central water of the Arabian Sea (20◦ ~11◦ N, 58◦ ~70◦ E), the eastern and western waters of the EEZ of the Maldives (9◦ N~5◦ S, 60◦ ~70◦ E; 6◦ N~5◦ S, 77◦ ~85◦ E), the high sea of the Bay of Bengal (18◦ ~12◦ N, 83◦ ~90◦ E), and the northeastern Indian Ocean (1◦ N~3◦ S, 88◦ ~94◦ E). Among these areas, fishing vessels are distributed throughout the year in the eastern waters of the Sensors 2024, 24, 781 16 of 19 Maldives’ EEZ, while in other areas, fishing vessel distributions change with seasons, which are related to factors such as geographical location, monsoon, and the different breeding, feeding, and migration periods of different catch targets. For instance, in the central water of the Arabian Sea, there are abundant catches of catch targets such as Indian mackerels (Rastrelliger kanagurta) and sardines. These species spawn in coastal waters during the southwest monsoon period and migrate to off-shore waters for feeding during the northeast monsoon period [55,56], which leads to fishing vessels in this water mostly operating during the northeast monsoon period. The eastern waters of theMaldives’ EEZ are abundant in tuna, including various species that spawn and migrate in different seasons. Some species, such as bigeye tuna (Thunnus obesus) and yellowfin tuna (Thunnus albacares), can be caught throughout the year [57]. This diversity contributes to fishing operations in this area throughout the year. The above speculations need to be further analyzed, combined with the biological characteristics of the catch targets. Understanding the migratory habits of the catch targets will also help reveal the fishing ground changes in high seas. 4.4. AIS Data Issues This study collected AIS data through website screenshots, providing only location information. In comparison with original AIS data, the obtained data have limitations, including lack of information, incomplete display of vessel points, and low resolution due to website permissions and display classification. In future research, we intend to improve data acquisition by using original AIS data, which contain more detailed information about fishing vessels, including vessel types, status, and specific operation times. 5. Conclusions This study investigated spatial–temporal distribution characteristics of fishing vessels in the northern Indian Ocean from March 2020 to February 2023 by using AIS data. Combined with simultaneous SST and Chl-a concentration data, spatial overlay maps of fishing operation areas and oceanographic factors were created. Additionally, monthly and interannual variations in the number of fishing vessels were analyzed. The following conclusions are drawn: (1) The northeast monsoon and southwest monsoon have significant impacts on the distribution of fishing vessels in the northern Indian Ocean. Fishing season occurs during the northeast monsoon period (October to March), while the transition and fishing moratorium take place during the southwest monsoon period (April to September). (2) Fishing vessels are mainly concentrated between 20◦ N and 6◦ S, extending from west of 90◦ E to the eastern coast of Africa. (3) The suitable SST contributed to high CPUEs in fishing operation areas during the northeast monsoon and fishing vessels are widely distributed in the areas with both mid-range and low-range Chl-a concentrations. (4) The monthly numbers of fishing vessels show seasonal variations, displaying a periodic pattern with an overall increasing trend. Due to the impact of the COVID-19 pandemic, the total number of fishing vessels decreased in 2020, but this was followed by a gradual recovery in the subsequent two years. (5) The optimum ranges of SST and Chl-a concentration for fishing operations are defined as 27.96 to 29.47 ◦ C for SST and 0.03 to 1.81 mg/m3 for Chl-a concentration. The application of AIS data can describe the characteristics of fishing vessel operations in detail, which could help fishery authorities understand the spatial–temporal distribution of fishing operations and improve or reasonably formulate fishery management policies. However, given the lack of catch data and the deficiency of collected data, this study still has limitations. In future research, there is an intention to explore the driving factors behind the spatial–temporal distribution of fishing vessels and identify key factors influencing this distribution with original AIS data and catch data. Sensors 2024, 24, 781 17 of 19 Author Contributions: Conceptualization, Q.X.; data curation, J.L. (Jie Li); formal analysis, J.L. (Jie Li) and Q.X.; funding acquisition, Q.X. and X.L.; investigation, J.L. (Jie Li) and Q.X.; methodology, J.L. (Jie Li) and Q.X.; project administration, Q.X.; resources, X.L. and Q.X.; software, J.L. (Jie Li) and J.L. (Jinghu Li); supervision, Q.X.; validation, J.L. (Jie Li) and Q.X.; visualization, J.L. (Jie Li) and Q.X.; writing—original draft preparation, J.L. (Jie Li); writing—review and editing, J.L. (Jie Li), Q.X. and M.A. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Chinese Academy of Science (133244KYSB20180029), the International Partnership Program of the Chinese Academy of Sciences, the Strategic Priority Research Program of the Chinese Academy of Sciences (Class A) (XDA19060501), and the Informatization Plan of the Chinese Academy of Sciences (CAS-WX2021SF-0408). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy protection reasons. Acknowledgments: The authors thank the anonymous reviewers for their valuable comments and suggestions. Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Tickler, D.; Meeuwig, J.J.; Palomares, M.L.; Pauly, D.; Zeller, D. 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Srivastava and Lacy Allergy, Asthma and Clinical Immunology 2014, 10(Suppl 2):A47 http://www.aacijournal.com/content/10/S2/A47 ALLERGY, ASTHMA & CLINICAL IMMUNOLOGY MEETING ABSTRACT Open Access Trafficking of TNF via recycling endosomes in neutrophils Nutan Srivastava*, Paige Lacy From Canadian Society of Allergy and Clinical Immunology Annual Scientific Meeting 2014 Ottawa, ON, Canada. 23-26 October 2014 Background Neutrophils are highly abundant innate immune cells that contribute to asphyxic episodes of acute asthma exacerbations, and secrete the proinflammatory cytokine tumour necrosis factor-a (TNF). Recycling endosomes (REs) are specialized secretory compartments that perform multiple functions including trafficking of cytokines to cell surfaces, although these are not characterized in neutrophils. Our objective is to identify trafficking components in neutrophils that may contribute to cytokine secretion. Methods The effect of bacterial lipopolysaccharide (LPS) stimulation on the trafficking of stored and newly synthesized TNF was determined by treatment of human peripheral blood neutrophils with or without cycloheximide. To visualize intracellular TNF, neutrophils were adhered to glass slides and treated with LPS for 1 h (10 ng/ml). Colocalization of TNFa in neutrophils was performed with transferrin-Alexa 488 and anti-VAMP3 (markers for RE), anti-CD63 and anti-CD66b a membrane markers for the primary and secondary granules in neutrophils, respectively. We also determined Rab5 and Rab7 colocalization with TNF (markers for early and late endosomes, respectively). Imaging was carried out by Deltavision OMX super resolution microscopy. Results LPS induced 30-40% TNF secretion from stored sources, with the remainder newly synthesized. We found that neutrophils possess REs as determined by transferrin uptake and VAMP3 labeling. TNF also colocalized with REs, primary and secondary granules as well as early and late endosomes, suggesting multiple sites of TNF storage and Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada trafficking in neutrophils. However, TNF only colocalized with VAMP3 around periphery of cells after 1 h stimulation with LPS, suggesting TLR4-induced TNF trafficking via REs. Conclusions The present study provides evidence that movement of TNF + VAMP-3+ vesicles towards the cell periphery in response to LPS. This suggests that neutrophils utilize REs for trafficking of TNF to the cell surface in response to TLR4 signalling. These findings contribute to our understanding of how neutrophils package, transport, and release cytokines. Published: 18 December 2014 doi:10.1186/1710-1492-10-S2-A47 Cite this article as: Srivastava and Lacy: Trafficking of TNF via recycling endosomes in neutrophils. Allergy, Asthma and Clinical Immunology 2014 10(Suppl 2):A47. 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 © 2014 Srivastava and Lacy; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 

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European Archives of Paediatric Dentistry (2021) 22:567–574 https://doi.org/10.1007/s40368-020-00591-1 ORIGINAL SCIENTIFIC ARTICLE The impact of dental caries and its treatment under general anaesthetic on children and their families R. Knapp1 · Z. Marshman1 · F. Gilchrist1 · H. Rodd1 Received: 1 July 2020 / Accepted: 18 November 2020 / Published online: 5 December 2020 © The Author(s) 2020 Abstract Objective To assess the impact of dental caries and treatment under general anaesthetic (GA) on the everyday lives of children and their families, using child-reported measures of quality of life (QoL) and oral health-related quality of life (OHRQoL). Method Participants, aged 5–16 years old having treatment for dental caries under GA, were recruited from new patient clinics at Charles Clifford Dental Hospital, Sheffield. OHRQoL was measured before and 3-months after treatment using the Caries Impacts and Experiences Questionnaire for Children (CARIES-QC). Overall QoL was measured using the Child Health Utility 9D (CHU9D). Parents/caregivers completed the Family Impact Scale (FIS). Results Eighty five parent–child dyads completed the study. There was statistically significant improvement in OHRQoL (mean interval score difference in CARIES-QC = 4.43, p < 0.001) and QoL (mean score difference in CHU9D = 2.48, p < 0.001) following treatment, with moderate to large effect sizes. There was statistically significant improvement in FIS scores (mean score difference = 5.48, p = 0.03). Conclusions Treatment under GA was associated with improvement in QoL and OHRQoL as reported by children, and reduced impacts on the family. This work highlights the importance of GA services in reducing the caries-related impacts experienced by children. Further work is needed investigate the impact of clinical, environmental and individual factors. Keywords Oral health · Quality of life · Paediatric dentistry · Caries Background Dental caries remains one of the most prevalent chronic diseases of childhood, affecting 60–90% of children worldwide. There is wide variation between with within countries, for example, in Europe between 20 and 90% of children aged six have dental caries (Petersen 2003). Although overall rates of dental caries are decreasing, these figures have shown inequalities within countries and across the region, with the highest burden of disease carried by those from lower socioeconomic groups (Petersen et al. 2005; Jakab 2016). In the UK, the most recent child dental health survey reported 31% of 5-year-old children had ‘obvious decay experience’, rising to nearly half by age eight (Steele et al. 2015). Many children with dental caries receive treatment * R. Knapp becky.knapp@sheffield.ac.uk 1 School of Clinical Dentistry, University of Sheffield, Sheffield, UK under general anaesthetic (GA) and in the UK, it remains the most common reason for a child to be admitted to hospital. In 2018/2019, in England alone, there were approximately 59,000 ‘Finished Consultant Episodes’ for children and adolescents (aged 0–19) admitted for dental extractions under GA (Public Health England 2020). These statistics are shocking when we consider that dental caries is an almost entirely preventable disease. While impacts of caries are well documented, what is less well understood is the subjective experience of children themselves. Several instruments have been developed to investigate the subjective impact of oral diseases, seeking to measure oral health-related quality of life (OHRQoL). A number of studies have demonstrated overall improvements in OHRQoL in children following treatment for dental caries under GA, however, most studies of child-OHRQoL have relied on proxy-reported measures rather than seeking the views of children themselves (Knapp et al. 2017). In addition, earlier studies have relied on generic measures of OHRQoL to measure change following treatment, may 13 Vol.:(0123456789) 568 not have been sensitive enough to capture caries-specific impacts (Guyatt et al. 1993). Therefore, the aim of this study was to assess the impact of dental caries and treatment under GA on the everyday lives of children and their families, using child-reported measures of OHRQoL and overall quality of life (QoL). Materials and methods Approval for this project was obtained from the South East Scotland NHS Research Ethics Committee (ref: 16/ SS/0187). A convenience sample of participants was recruited from new patient clinics on the Paediatric Clinic, Charles Clifford Dental Hospital, Sheffield, UK between January 2017 and January 2019. A sample size calculation revealed that 84 participants would be needed to detect an effect size of 0.4 (i.e., medium effect) at 5% level of significance and 95% power. All children, and their parents, who met the eligibility criteria for the study, were invited to participate. Inclusion criteria were: children aged 5–16 years; with active dental caries; who are otherwise medically fit and well; children and parents both able to understand spoken English, i.e., able to understand and undertake the research with support. Potential participants were excluded if they had caries in conjunction with any other dental conditions, e.g., trauma or dental anomaly, such as molar incisor hypomineralisation, as due to the numbers of participants involved there would not be sufficient data to account for this as a potential confounding factor in the results. This study employed a pre-test/post-test design. Children were given age-appropriate information sheets at their assessment visit and, if they wished to participate, were asked to complete consent forms and questionnaires at the following time points: pre-test at their new patient appointment, and post-test, 3 months following treatment under GA, self-completed at home and returned by post. Children were asked to complete the questionnaire themselves, but younger children were given assistance if needed, e.g., having the questions read to them, but with no additional guidance on the answers. Parents were asked to complete a separate consent form and questionnaire at the same time points. Once participants had completed the study, they were given a £10 ‘Love to Shop’ gift voucher as a thank you for their time. Where parents did not agree to participate, the parent/child dyad was excluded from the study. Measures used The child questionnaire consisted of two sections: the Caries Impacts and Experiences Questionnaire for Children 13 European Archives of Paediatric Dentistry (2021) 22:567–574 (CARIES-QC) designed to measure OHRQoL and the Child Health Utility 9D (CHU9D) which measures overall QoL. CARIES-QC had previously been validated for use in this population. It is a caries-specific questionnaire and was designed for completion by children themselves. It contains 12 items with a 3-point response format, where children rate whether they are affected ‘not at all’, ‘a bit’ or ‘a lot’, with respective scores of 0, 1 and 2. A global question asks children to rate how much of a problem their teeth are overall, on the same 3-point scale. Total raw scores range from 0 to 24, where higher scores indicate worse OHRQoL. To calculate change following treatment, raw scores are converted to an interval scale score, which allows for the more accurate calculation of change at all points on the scale (Gilchrist et al. 2018). The CHU-9D consists of nine items, each with five ordinal responses (scored 1–5) that assess the child’s functioning across domains, such as worry, pain, tiredness, and daily routine. Overall scores range from 9 to 45, where increasing score implies greater impact on QoL (Stevens and Ratcliffe 2012). The parent/caregiver questionnaire consisted of the Family Impact Scale (FIS) and additional questions regarding the child’s dental history. The FIS consists of 14 items, with 5-point Likert scale to record the frequency of each impact scored as follows: ‘Never’ = 0; ‘once or twice’ = 1; ‘sometimes’ = 2; ‘often’ = 3; ‘every day or almost every day’ = 4. A ‘don’t know’ response is also possible and scored as 0. This instrument is validated for use in this population (de Souza et al. 2016). Total FIS score ranges from 0 to 56, with higher scores representing greater family impact. Subscale scores were obtained by summing subsets of items within the categories of parental/family activity, parental emotions and family conflict. Global transition questions were also included. In terms of additional questions, parents were asked if the child had received antibiotics for their dental problem, and if so, how many courses, as well as whether the child or a sibling had received a dental GA previously. Information was obtained by parents in the questionnaire as it was not always available from the referral letter or patient record. Demographic and clinical data were also collected, from the patient record. Individual characteristics included the age of the child, in years, and their ethnicity, recorded as ‘White British’ or ‘Black and Minority Ethnic’ (BME). Deprivation, which was assessed using a composite measure of area-based deprivation, the Index of Multiple Deprivation 2015 (IMD) score derived from their house postcode, was recorded. Both the overall IMD rank score and quintile were recorded, with quintile 1 being the most advantaged, and quintile 5 being the most disadvantaged quintile. The number of carious teeth was recorded based on clinical examination at their new patient appointment. This usually included European Archives of Paediatric Dentistry (2021) 22:567–574 caries recorded following radiographic assessment, therefore early carious lesions as well as cavitated lesions, unless the child was unable to tolerate radiographs being taken. Data were also collected on whether the child had anterior caries and whether they reported pain at the assessment, as well as if there were any safeguarding concerns in place. Safeguarding concerns were indicated in the patient record by the child’s placement on a historic or current care protection plan, whether they had a paediatric liaison letter or whether there was social care involvement (a named social worker, as detailed in the new patient proforma). Data analysis Data were entered into an electronic database (IBM SPSS Statistics, version 24) and descriptive statistics reported. All statistical analysis results were considered significant at p < 0.05. Change following treatment Total child-OHRQoL (CARIES-QC) and QoL (CHU9D) scores at baseline and 3 months following treatment were calculated and change in scores analysed using the Mann–Whitney test. Change scores were calculated by subtracting follow-up scores from baseline scores, so a positive change indicated improvement in QoL. Cohen’s d effect sizes were calculated for change scores to assess the magnitude of change, with effect sizes of 0.2, 0.2–0.7 and greater than 0.7 representing small, moderate and large magnitudes of change, respectively (Sawilowsky 2009). The minimal important difference (MID), i.e., the smallest difference in the score which is considered clinically meaningful and which patients perceive as beneficial (Masood et al. 2014), of the CARIES-QC and FIS scores, was calculated using the mean change scores of participants who reported ‘improvement’ on the global rating. Psychometric properties Internal consistency was assessed using the Cronbach’s alpha test. By convention, the alpha cut-off value of 0.70 or higher was considered acceptable (Kline 1999). The longitudinal construct validity and responsiveness of CARIES-QC and FIS were assessed by comparing mean change scores for each measure with the global transition question responses. As CHU9D did not include a global question, it was not tested at this point. 569 Results Sample characteristics A total of 273 patients were approached to participate in the study between January 2017 and January 2019, of which 106 declined to participate (response rate = 61.2%). Of the 167 who consented to take part, 82 participants were subsequently lost to follow-up. In total, therefore, 85 parent–child dyads completed the study (completion rate = 50.9%). The age of children who completed the study ranged from 5 to 11 years (mean = 6.5 ± 1.5). Nearly three-quarters (n = 62, 72.9%) of children were from the most deprived areas of England. Most children were White British (n = 62, 72.9%). There was no significant difference in these demographic variables between those who completed the study and those who did not (Table 1). Clinical data from the patient record showed that the number of carious teeth ranged from 1 to 15 (mean = 6.6 ± 2.9). Anterior caries was present in 12% (n = 10) of the participants. Pain was reported at initial assessment by 70.6% (n = 60) of children. In addition, 8 (9.4%) children had safeguarding concerns in place. Antibiotics had been received by 43.5% (n = 37) of children prior to their assessment visit. Over a quarter (27%, n = 23) of children had a sibling who had previously received treatment under GA, while 2.4% (n = 2) of children had previously had dental treatment under GA themselves. Quality‑of‑life results The total scores for each measure, before and three months after treatment, are given in Table 2. Effect sizes for the change in score are also given. There was a statistically significant improvement in OHRQoL (mean score difference in CARIES-QC = 4.43 ± 4.92, p < 0.001) and QoL (mean score difference in CHU9D = 2.48 ± 5.29, p < 0.001) following treatment, with moderate to large effect sizes. At baseline, the main OHRQoL impacts reported by children were food getting stuck in their teeth (91%), having to eat on one side (64%) and their teeth causing them to cry (56%). All these impacts reduced following treatment under GA (Table 3). Data from the CARIES-QC global question revealed that, before treatment, 58% of the children said their teeth were a problem for them, and this figure reduced to 21% three months following treatment. Overall, 92% of children reported that their teeth were ‘better’ following treatment. The remaining 8% reported their teeth were ‘the same’ as before treatment. The most common impacts on the family of these children before treatment were parents feeling upset (75.3%, 13 570 European Archives of Paediatric Dentistry (2021) 22:567–574 Table 1  Comparison of participants’ demographic characteristics at baseline, of those followed up and those lost to follow-up Variable All (n = 167) Age, years Range 5–14 Mean (± SD) 6.70 (± 1.69) Gender Male 79 (47.3%) Female 88 (52.7%) Ethnicity White British 121 (72.5%) BME 46 (27.5%) Deprivation (based on IMD score) Least deprived 10 (6.0%) Less deprived 19 (11.4%) Average 16 (9.6%) More deprived 36 (21.5%) Most deprived 86 (51.5%) Safeguarding concern No 138 (82.6%) Yes 20 (12.0%) Data missing 9 (5.4%) Followed up (n = 85) Lost to follow-up (n = 82) p-value 5–11 6.49 (± 1.53) 5–14 6.91 (± 1.83) 0.14 38 (44.7%) 47 (55.3%) 41 (50.0%) 41 (50.0%) 0.49 62 (72.9%) 23 (27.1%) 59 (72.0%) 23 (28.0%) 0.89 6 (7.1%) 10 (11.8%) 7 (8.2%) 14 (16.5%) 48 (56.5%) 4 (4.9%) 9 (11.0%) 9 (11.0%) 22 (26.8%) 38 (46.3%) 0.46 77 (90.6%) 8 (9.4%) 0 61 (74.4%) 12 (14.6%) 9 (11.0%) 0.19 Numbers, with percentages in brackets, are given unless otherwise stated SD standard deviation; p−values are for comparisons between the followed−up and lost to follow−up groups. As the data were not normally distributed, the Mann–Whitney U test was used to test for significant difference between the groups. Pearson’s Chi−squared test was used to test for difference in categorical variables. There were no statistically significant results. BME black or minority ethnic group Table 2  Mean overall scores at baseline and follow-up, with effect sizes Measure CARIES-QC interval CHU9D FIS Baseline 8.99 ± 4.29 (0–19.96) 13.58 ± 4.96 (9–31) 9.21 ± 7.31 (0–35) Follow-up 4.47 ± 5.58 (0–16.17) 11.09 ± 3.07 (9–24) 7.02 ± 6.40 (0–28) Change p-value Cohen’s d effect size Effect size description 4.43 ± 4.92 (− 8.63–16.92) < 0.001* 0.91 Large 2.48 ± 5.29 (− 14–20) 2.19 ± 7.84 (− 17–24) < 0.001* 0.03* 0.60 0.32 Moderate Moderate Scores show mean ± SD (range). p−values are for Wilcoxon test for difference between baseline and follow−up scores *Statistically significant result (p<0.05) n = 64), feeling guilty (75.3%, n = 63), having disturbed sleep (58.8%, n = 50) and the child requiring more attention (48.2%, n = 41). Nearly half the parents had to take time off work (45.9%, n = 39). Following treatment, the numbers of parents reporting each of these impacts reduced, albeit to different degrees. The biggest effects were seen in fewer parents having sleep disrupted and fewer feeling upset and guilty. There was a statistically significant change in overall FIS score following treatment (mean score difference = 2.19 ± 7.84, p = 0.03), however, only the ‘parental and family activities’ domain saw a statistically significant change in score between baseline and follow-up. 13 Responses to the global questions revealed that nearly all the parents (95%, n = 81) rated their child’s oral health as improved at follow-up. Approximately three-quarters of parents felt the overall quality of life of their children had improved (74%, n = 63), and none felt it had worsened. Just over half the parents (n = 46) reported that the impact on the family had improved, with the remaining parents reporting it had stayed the same. The minimally important difference was 4.68 for CARIES-QC interval score and 1.65 for FIS. Overall, therefore, 40 participants (47.1%) exceeded the MID for CARIES-QC and 24 (28.2%) did so for the FIS. European Archives of Paediatric Dentistry (2021) 22:567–574 Table 3  Number and proportion of children responding positively (‘a bit’ or ‘a lot’) to each item at baseline and follow-up 571 Item Number (%) at baseline Number (%) at 3-months follow-up Reduction in % of children reporting this item at followup Food stuck Feel cross Cried Eat more carefully Eating on one side Teeth hurt Hard to eat some foods Annoyed Eat more slowly Kept awake Hurt when brushing Hard to do schoolwork 73 (85.9%) 61 (71.8%) 61 (71.8%) 52 (61.2%) 50 (58.8%) 48 (56.5%) 47 (55.3%) 45 (52.9%) 35 (41.2%) 33 (38.8%) 33 (38.8%) 17 (20.0%) 50 (58.8%) 13 (15.3%) 27 (31.8%) 22 (25.9%) 32 (37.6%) 15 (17.6%) 27 (31.8%) 19 (22.4%) 17 (20.0%) 3 (3.5%) 14 (16.5%) 3 (3.5%) 31.5% 78.7% 55.7% 60.0% 36.0% 68.8% 42.6% 57.8% 51.4% 91.0% 57.6% 82.4% Table 4  Mean change in quality of life scores by response to the global transition question Response to GTJ Improved Stayed the same Got worse CARIES-QC Interval FIS Number (%) Change score p-value Number (%) Change score p-value 78 (91.8%) 7 (8.2%) 0 4.68 ± 4.81 (− 7.8–16.92) 1.69 ± 5.61 (− 8.63–6.69) n/a < 0.001* 0.49 n/a 46 (54.1%) 39 (45.9%) 0 1.65 ± 9.23 (− 17–24) 2.82 ± 5.83 (− 7–20) n/a 0.46 0.009 n/a CARIES−QC interval scores show mean ± SD (range). p−values are for Wilcoxon test for difference between baseline and follow−up scores for each group *Statistically significant result between scores at baseline and follow−up (p<0.05) Psychometric properties of the measures used CARIES-QC demonstrated excellent internal consistency (α = 0.9), CHU-9D and FIS both demonstrated good internal consistency (α = 0.8) overall. Table 4 shows the results of longitudinal validity and responsiveness testing. CARIES-QC demonstrated good longitudinal validity and responsiveness overall; however, FIS demonstrated poor longitudinal validity and responsiveness. Discussion This study aimed to investigate the effect of treatment for dental caries under GA on the everyday lives of children and their families. In contrast to previous studies, CARIES-QC has provided insights into the aspects of a child’s daily life which are most improved following treatment, from their own perspective rather than relying on a proxy report. The other advantage to the use of CARIESQC in this study, as the only disease-specific measure of OHRQoL available, is that it may have identified impacts which are specific to dental caries. The results from this study have demonstrated that dental caries has a significant impact on children and their families. The main impacts of caries reported by children were related to eating and their teeth causing them to cry. The study has also demonstrated wider effects on the family and society, for example, parents feeling guilty and days lost at work. The findings, in relation to the sample characteristics and quality-of-life impacts are discussed in more detail below. Sample characteristics The participants in this study had high levels of caries experience. The most recent survey of 5-year-old children in England found that the mean dmft for Sheffield children who had caries experience was 3.5 (Public Health England 2017). The dmft of children included in this study was considerably higher than this at 6.9, although the figure is similar to that 13 572 in other studies investigating the impact of treatment under GA in children (e.g., Gilchrist et al. 2018). This study clearly highlighted ongoing inequalities in children’s oral health as nearly three-quarters of those requiring treatment under GA in this sample were living in the most deprived areas of England. These findings support those in other studies where children receiving treatment under GA tended to be from socially deprived backgrounds (Hariharan et al. 2017). An important finding in this study was that over a quarter of children (27%) had a sibling who had previously received dental treatment under GA, highlighting the need for targeted oral health promotion and caries prevention programmes to help reduce inequalities in the burden of disease in deprived populations. Nationwide interventions have been used elsewhere in the UK with success in reducing inequalities, for example, ‘Childsmile’ in Scotland (McMahon et al. 2011) and ‘Designed to Smile’ in Wales (Morgan 2018). There is a need also to ensure there is sufficient remuneration for dentists to carry out preventative activity and to provide restorative care (Watt et al. 2019). Additionally, there were several children in this study (9%, n = 8) who had safeguarding concerns in place. Perhaps more worryingly, a greater number (15%, n = 12) of children with safeguarding concerns in place were lost to follow-up, some of whom will have done so because they failed to return for further treatment. These children would have been processed through the departmental protocol for children who are not brought to appointments, which would have included communication with the safeguarding teams to ensure they were seen again at a later stage. However, this process means that there would have been a delay in these children receiving the treatment they required. Failure or delay in seeking dental care, including for dental caries, is a cause for concern, all the more so in the UK where child dental care is available free-of-charge on the NHS and cost is not a barrier to access. It is only more recently that missed appointments for dentistry have been considered from a safeguarding perspective (Harris 2018). This study highlights the impacts of dental caries which will be experienced by children who have a delay in receiving treatment under GA, either from not being brought to appointments, or where there is a lack of GA service provision resulting in long waiting times for treatment, or where GA services are reduced for other reasons, as has been the case during the recent COVID-19 pandemic. The results from the study also show that significant numbers of children (43.5%) had received antibiotics prior to their initial assessment for a GA. These findings are concerning in the light of growing antibiotic resistance. It brings into question the prescribing practices of dentists, and whether these antibiotics are always indicated. It may be that antibiotics are viewed as a course of ‘treatment’ in the interim period from referral to assessment, where a dentist 13 European Archives of Paediatric Dentistry (2021) 22:567–574 is unable to carry out treatment in the dental chair. Indeed, a survey of prescribing practices in the North of England found that there was only evidence of spreading infection or systemic involvement in approximately half of the cases, and that other reasons, such as patient expectations, time pressure and lack of co-operation, were impacting on the decision to prescribe antimicrobials (Sturrock et al. 2018). Although antibiotic prescribing in dentistry has reduced in recent years, 5.2% of all antibiotics are prescribed in dentistry (Public Health England, 2018), suggesting more needs to be done. Common approaches with medical colleagues to educate the wider public about the need to reduce antibiotic use and why antibiotics might not be appropriate is advocated, alongside support for dentists to have the confidence and time to do the right thing. This has implications for practice, such as ensuring urgent care appointments are available and appropriately remunerated to allow time for appropriate treatment to be carried out. Quality‑of‑life findings There were statistically significant improvements in overall child OHRQoL scores (p < 0.001), child- HRQoL scores (p < 0.001) and in FIS scores (p = 0.03), 3 months following treatment under GA. The change in scores represented moderate to large effect sizes. All individual impacts were reduced following treatment, albeit to varying degrees. These results suggest that treatment under GA results in significant improvements in the OHRQoL and overall QoL of children, three months following treatment. The negative impact on the everyday lives of the families of these children was also significantly reduced following treatment. CARIES-QC demonstrated good overall internal consistency (Cronbach alpha = 0.9). This value is slightly higher than that found in work by Foster-Page and colleagues (2019), who obtained an alpha of 0.8; and adds to the evidence that CARIE-QC has good overall internal consistency. CARIES-QC also good demonstrated longitudinal construct validity and responsiveness, supporting its use to evaluate change following treatment for dental caries in children. Interestingly, the large effect sizes seen for change in CARIES-QC scores in this study were greater than those previously reported in the study by Foster-Page and team (2019), who found moderate effect sizes. This may be because all the child participants in this study were requiring treatment under GA, which may reflect a greater treatment need than in the other study populations. In contrast, the FIS demonstrated poor validity and responsiveness in this population, and variable internal consistency. This may be because it is a generic rather than caries-specific measure, unable to detect changes in caries-related impacts on the family. Further psychometric testing of all measures, especially in European Archives of Paediatric Dentistry (2021) 22:567–574 larger samples is warranted. Factor and Rasch analysis may be helpful to establish which questions may not be performing as well. Limitations It is worth noting some limitations of this study. While the completion rate of 50.9% in this study was comparable to similar studies (Martins-Junior et al. 2017), there was still a significant loss to follow-up. Comparison between those lost to follow-up and those completing the study confirmed that there was no difference in baseline characteristics between the two groups; however, future work should explore ways of trying to improve retention to such studies. It is well documented that individuals from lowincome families are less likely to participate in research studies than those from higher-income backgrounds (Heinrichs et al. 2005). While the reasons for this are complex, this work highlights the need to consider how best to reach this group of patients. The inconvenience of completing and returning a paper questionnaire could be considerable. Although participants were given a gift voucher to try and compensate them for their time, it may have been that this was not seen as sufficient. A systematic review of strategies to improve retention in clinical trials found that the most effective method for increasing response rates to postal questionnaires was to give a monetary incentive, and the higher the incentive the higher the response rates (Brueton et al. 2014). It might be possible in future to make use of other methods, for example, by including other participatory approaches, such as drawings or activities, to encourage the involvement of children (Marshman and Hall 2008). In addition, children were only able to participate if their parents consented too, reducing the number of child participants. If possible, future work should consider child consent practices and the possibility of allowing children to participate even if their parents do not wish to. One of the exclusion criteria was to exclude those with other dental conditions in conjunction with caries, as it was felt this could impact on OHRQoL scores. Ideally, future studies would seek to recruit all individuals with caries, but recruit in sufficient numbers that analysis could be made to determine if these additional dental conditions had a significant effect on OHRQoL outcomes. Another limitation was the requirement for individuals to understand spoken English to allow completion of the questionnaire with support. This decision was made as, at the time of commencing the study, CARIES-QC was only validated for use in an English-speaking population. Translation may have altered the meaning of the questions and a translator may not have always been available. This could 573 have led to selection bias in the sample, and therefore future work should seek to overcome this language restriction. In conclusion, this research contributes to the field as it includes a disease-specific, child-reported measure to examine changes in OHRQoL following treatment for caries under GA. It has demonstrated that dental treatment under GA is associated with significant improvement in the OHRQoL and QoL of children, and in the impact on their families. Future work should be carried out to investigate these findings in more detail, taking into account individual, clinical and environmental factors which may impact on the results. The study has added to the evidence which shows all three measures demonstrate good internal consistency. This research also provided evidence of the usefulness of CARIES-QC but raised some questions regarding the usefulness of the FIS, in longitudinal research in similar populations. Conclusion Treatment under GA was associated with significant improvement in QoL and OHRQoL as reported by both children and their parents. This work highlights the importance of dental care under GA for children with dental caries, which has implications for service planning and resource allocation. Treatment under GA requires significant theatre time and associated resources for treating dental caries in children, and this paper provides further evidence for both the impacts of caries on children and their families prior to treatment and the effectiveness of treatment in reducing those impacts. Further work is needed investigate the impact of clinical, environmental and individual factors on quality-of-life outcomes. Acknowledgements Thanks goes to all the children and families who gave up their time to participate in the study. Our gratitude goes out to the staff in the Paediatric Dental Clinic at the Charles Clifford Dental Hospital, whose help in the running of this project was greatly appreciated. Thanks are also given to the David Locker Scholarship foundation, for the award of the scholarship which enabled this work to be completed. Funding This study was funded by a PAPOR PhD Scholarship from the School of Clinical Dentistry, Sheffield, UK and the David Locker Scholarship. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. 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 13 574 were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. 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